TECHNICAL FIELD
[0001] This invention relates to nucleic acid and amino acid sequences of synthetases and
to the use of these sequences in the diagnosis, treatment, and prevention of immune,
neuronal, and reproductive disorders, and cell proliferative disorders including cancer.
BACKGROUND OF THE INVENTION
[0002] A large number of cellular biosynthetic intermediary metabolism processes involve
intermolecular transfer of carbon atom-containing substrates (carbon substrates).
Examples of such reactions include the tricarboxylic acid cycle, synthesis of fatty
acids and long-chain phospholipids, synthesis of alcohols and aldehydes, synthesis
of intermediary metabolites, and reactions involved in the amino acid degradation
pathways. Many of these reactions are catalyzed by synthetases (also called ligases),
which catalyze the formation of a bond between two substrate molecules. Some of these
reactions require input of energy, usually in the form of conversion of ATP to either
ADP or AMP and pyrophosphate. Synthetases are named for the products of the reaction
they catalyze and are involved in such processes as metabolism and the synthesis of
macromolecules.
Ligases forming carbon-oxygen bonds
[0003] Proteins make up more than half of the total dry mass of a cell. The synthesis of
proteins is central to cell maintenance, growth, and development. Synthesis occurs
on ribosomes and depends on the cooperative interaction of several classes of RNA
molecules. The process begins with transcription of the genetic code contained within
the DNA to form messenger RNA (mRNA). The mRNA moves in steps through a ribosome and
the nucleotide sequence of the mRNA is translated into a corresponding sequence of
amino acids to construct a distinct protein chain.
[0004] The aminoacyl-transfer RNA (tRNA) synthetases are important RNA-associated enzymes
with roles in translation. Protein biosynthesis depends on each amino acid forming
a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible
for the activation and correct attachment of an amino acid with its cognate tRNA.
The 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes,
and each class is characterized by a distinctive topology of the catalytic domain.
Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman
'fold'. Class II enzymes contain a central catalytic domain, which consists of a seven-stranded
antiparallel β-sheet motif, as well as N- and C- terminal regulatory domains. Class
II enzymes are separated into two groups based on the heterodimeric or homodimeric
structure of the enzyme; the latter group is further subdivided by the structure of
the N- and C-terminal regulatory domains (Hartlein, M. and Cusack, S. (1995) J. Mol.
Evol. 40:519-530). Autoantibodies against aminoacyl-tRNAs are generated by patients
with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial
lung disease (ILD). These antibodies appear to be generated in response to viral infection,
and coxsackie virus has been used to induce experimental viral myositis in animals
(Friedman, A.W. et al. (1996) Semin. Arthritis Rheum. 26:459-467). A synthetase homolog
has been shown to be expressed in chronic myeloid leukemia (CML). A phenylalanine-tRNA
synthetase homolog has been found to be tumor-selective and expressed in a cell cycle
stage- and differentiation-dependent fashion in an acute-phase human CML cell line
(Sen, S. et al. (1997) Proc. Natl. Acad. Sci.USA 94:6164-6169).
Ligases forming carbon-sulfur bonds (Acid-thiol ligases)
[0005] In many cases, a carbon substrate is derived from a small molecule containing at
least two carbon atoms. The carbon substrate is often covalently bound to a larger
molecule which acts as a carbon substrate carrier molecule within the cell. In the
biosynthetic mechanisms described above, the carrier molecule is coenzyme A. Coenzyme
A (CoA) is structurally related to derivatives of the nucleotide ADP and consists
of 4'-phosphopantetheine linked via a phosphodiester bond to the alpha phosphate group
of adenosine 3',5'-bisphosphate. The terminal thiol group of 4'-phosphopantetheine
acts as the site for carbon substrate bond formation. The predominant carbon substrates
which utilize CoA as a carrier molecule during biosynthesis and intermediary metabolism
in the cell are acetyl, succinyl, and propionyl moieties, collectively referred to
as acyl groups. Other carbon substrates include enoyl lipid, which acts as a fatty
acid oxidation intermediate, and carnitine, which acts as an acetyl-CoA flux regulator/
mitochondrial acyl group transfer protein. Acyl-CoA and acetyl-CoA are synthesized
in the cell by acyl-CoA synthetase and acetyl-CoA synthetase, respectively.
[0006] Activation of fatty acids is mediated by at least three forms of acyl-CoA synthetase
activity: i) acetyl-CoA synthetase, which activates acetate and several other low
molecular weight carboxylic acids and is found in muscle mitochondria and the cytosol
of other tissues; ii) medium-chain acyl-CoA synthetase, which activates fatty acids
containing between four and eleven carbon atoms (predominantly from dietary sources),
and is present only in liver mitochondria; and iii) acyl CoA synthetase, which is
specific for long chain fatty acids with between six and twenty carbon atoms, and
is found in microsomes and the mitochondria. Proteins associated with acyl-CoA synthetase
activity have been identified from many sources including bacteria, yeast, plants,
mouse, and man. The activity of acyl-CoA synthetase may be modulated by phosphorylation
of the enzyme by cAMP-dependent protein kinase. The COL4A5 (collagen, type IV, alpha-5)
chromosomal region found deleted in 2 patients with Alport syndrome, elliptocytosis,
and mental retardation (Piccini. M. et al, (1998) Genomics 47: 350-358) is contiguous
with the region containing long-chain acyl-CoA synthetase 4 (FACL4). Therefore, it
has been suggested (Piccini supra) that the absence of FACL4 may be involved in the
development of mental retardation and other phenotypes associated with Alport syndrome
in these patients.
Ligases forming carbon-nitrogen bonds
[0007] A key representative of the amide synthases is the enzyme glutamine synthetase (glutamate-ammonia
ligase) that catalyzes the amination of glutamic acid to glutamine by ammonia using
the energy of ATP hydrolysis. Glutamine is the primary source for the amino group
in various amide transfer reactions involved in de novo pyrimidine nucleotide synthesis
and in purine and pyrimidine ribonucleotide interconversions, as well as the conversion
of aspartate to asparagine. Overexpression of glutamine synthetase has been observed
in primary liver cancer (Christa. L. et al. (1994) Gastroent. 106:1312-1320).
[0008] Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and enzyme
complexes that participate in the de novo pathways to purine and pyrimidine biosynthesis.
Because these pathways are critical to the synthesis of nucleotides for replication
of both RNA and DNA, many of these enzymes have been the targets of clinical agents
for the treatment of cell proliferative disorders such as cancer and infectious diseases.
[0009] Purine biosynthesis occurs de novo from the amino acids glycine and glutamine, and
other small molecules. Three of the key reactions in this process are catalyzed by
a trifunctional enzyme composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole
ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART).
Together these three enzymes combine ribosylamine phosphate with glycine to yield
phosphoribosyl aminoimidazole, a precursor to both adenylate and guanylate nucleotides.
This trifunctional protein has been implicated in the pathology of Downs syndrome
(Aimi, J. et al. ( 1990) Nucleic Acid Res. 18:6665-6672). Adenylosuccinate synthetase
catalyzes a later step in purine biosynthesis that converts inosinic acid to adenylosuccinate,
a key step on the path to ATP synthesis. This enzyme is also similar to another carbon-nitrogen
ligase, argininosuccinate synthetase, that catalyzes a similar reaction in the urea
cycle (Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
[0010] Like the de novo biosynthesis of purines, de novo synthesis of the pyrimidine nucleotides
uridylate and cytidylate also arises from a common precursor, in this instance the
nucleotide orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
Again a trifunctional enzyme comprising three carbon-nitrogen ligases plays a key
role in the process. In this case the enzymes aspartate transcarbamylase (ATCase),
carbamyl phosphate synthetase II, and dihydroorotase (DHOase) are encoded by a single
gene called CAD. Together these three enzymes combine the initial reactants in pyrimidine
biosynthesis, glutamine, CO
2, and ATP to form dihydroorotate, the precursor to orotate and orotidylate (Iwahana,
H. et al. (1996) Biochem. Biophys. Res. Commun. 219:249-255). Further steps then lead
to the synthesis of uridine nucleotides from orotidylate. Cytidine nucleotides are
derived from uridine-5'-triphosphate (UTP) by the amidation of UTP using glutamine
as the amino donor and the enzyme CTP synthetase. Regulatory mutations in the human
CTP synthetase are believed to confer multi-drug resistance to agents widely used
in cancer therapy (Yamauchi, M. et al. (1990) EMBO J. 9:2095-2099).
Ligases forming carbon-carbon bonds
[0011] Ligases in this group are represented by the carboxylases acetyl-CoA carboxylase
and pyruvate carboxylase. Acetyl-CoA carboxylase is a complex which includes a biotin
carboxyl carrier protein, biotin carboxylase, and a carboxyl transferase made up of
two alpha and two beta subunits. This complex catalyzes the carboxylation of Acetyl-CoA
from CO
2 and H
2O using the energy of ATP hydrolysis (PRINTS document PR01069). Acetyl-CoA carboxylase
is the rate-limiting step in the biogenesis of long-chain fatty acids. Two isoforms
of Acetyl-CoA carboxylase, types I and types II, are expressed in humans in a tissue-specific
manner (Ha, J. et al. (1994) Eur. J. Biochem. 219:297-306). Pyruvate carboxylase is
a nuclear-encoded mitochondrial enzyme that catalyzes the conversion of pyruvate to
oxaloacetate, a key intermediate in the citric acid cycle.
Ligases forming phosphoric ester bonds
[0012] Ligases in this group are represented by the DNA ligases involved in both DNA replication
and repair. DNA ligases seal phosphodiester bonds between two adjacent nucleotides
in a DNA chain using the energy from ATP hydrolysis to first activate the free 5'-phosphate
of one nucleotide and then react it with the 3'-OH group of the adjacent nucleotide.
This resealing reaction is used in both DNA replication to join small DNA fragments
called "Okazaki" fragments that are transiently formed in the process of replicating
new DNA, and in DNA repair. DNA repair is the process by which accidental base changes,
such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation
of DNA, are corrected before replication or transcription of the DNA can occur. Bloom's
syndrome is an inherited human disease in which individuals are partially deficient
in DNA ligation and consequently have an increased incidence of cancer (Alberts. B.
et al. (1994)
The Molecular Biology of the Cell, Garland Publishing Inc., New York, NY, p. 247).
[0013] The discovery of new synthetases and the polynucleotides encoding them satisfies
a need in the art by providing new compositions which are useful in the diagnosis,
prevention, and treatment of immune, neuronal, and reproductive disorders, and cell
proliferative disorders including cancer.
SUMMARY OF THE INVENTION
[0014] The invention features purified polypeptides, human synthetases, referred to collectively
as "SYNT' and individually as "SYNT-1," "SYNT-2," "SYNT-3," "SYNT-4,""SYNT-5," "SYNT-6,"
"SYNT-7," "SYNT-8," "SYNT-9,""SYNT-10," "SYNT-11," "SYNT- 12," "SYNT-13," "SYNT-14,"
and "SYNT-15." In one aspect, the invention provides an isolated polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid sequence
selected from the group consisting of SEQ ID NO: 1-15. b) a naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ-ID NO:1-15, and d)
an immunogenic fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-15. In one alternative, the invention provides an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:1-15.
[0015] The invention further provides an isolated polynucleotide encoding a polypeptide
comprising an amino acid sequence selected from the group consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-15,
and d) an immunogenic fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-15. In one alternative, the polynucleotide encodes a polypeptide selected
from the group consisting of SEQ ID NO:1-15. In another alternative, the polynucleotide
is selected from the group consisting of SEQ ID NO:16-30.
[0016] Additionally, the invention provides a recombinant polynucleotide comprising a promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising an
amino acid sequence selected from the group consisting of a) an amino acid sequence
selected from the group consisting of SEQ ID NO:1-15, b) a naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence selected
from the group consisting of SEQ ID NO: 1-15, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, and d)
an immunogenic fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-15. In one alternative, the invention provides a cell transformed with
the recombinant polynucleotide. In another alternative, the invention provides a transgenic
organism comprising the recombinant polynucleotide.
[0017] The invention also provides a method for producing a polypeptide comprising an amino
acid sequence selected from the group consisting of a) an amino acid sequence selected
from the group consisting of SEQ ID NO:1-15, b) a naturally occurring amino acid sequence
having at least 90% sequence identity to an amino acid sequence selected from the
group consisting of SEQ ID NO:1-15, c) a biologically active fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.
The method comprises a) culturing a cell under conditions suitable for expression
of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide
comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide,
and b) recovering the polypeptide so expressed.
[0018] Additionally, the invention provides an isolated antibody which specifically binds
to a polypeptide comprising an amino acid sequence selected from the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-15,
b) a naturally occurring amino acid sequence having at least 90% sequence identity
to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c)
a biologically active fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence selected
from the group consisting of SEQ ID NO:1-15.
[0019] The invention further provides an isolated polynucleotide comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of SEQ ID NO:16-30, b) a naturally occurring polynucleotide
sequence having at least 90% sequence identity to a polynucleotide sequence selected
from the group consisting of SEQ ID NO:16-30, c) a polynucleotide sequence complementary
to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent
of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous
nucleotides.
[0020] Additionally, the invention provides a method for detecting a target polynucleotide
in a sample, said target polynucleotide having a sequence of a polynucleotide comprising
a polynucleotide sequence selected from the group consisting of a) a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 16-30, b) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a polynucleotide
sequence selected from the group consisting of SEQ ID NO:16-30, c) a polynucleotide
sequence complementary to a), d) a polynucleotide sequence complementary to b), and
e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with
a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary
to said target polynucleotide in the sample, and which probe specifically hybridizes
to said target polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or fragments thereof, and
b) detecting the presence or absence of said hybridization complex, and optionally,
if present, the amount thereof. In one alternative, the probe comprises at least 60
contiguous nucleotides.
[0021] The invention further provides a method for detecting a target polynucleotide in
a sample, said target polynucleotide having a sequence of a polynucleotide comprising
a polynucleotide sequence selected from the group consisting of a) a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 16-30, b) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 16-30, c) a polynucleotide
sequence complementary to a), d) a polynucleotide sequence complementary to b), and
e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide
or fragment thereof using polymerase chain reaction amplification, and b) detecting
the presence or absence of said amplified target polynucleotide or fragment thereof,
and, optionally, if present, the amount thereof.
[0022] The invention further provides a pharmaceutical composition comprising an effective
amount of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15, b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15,
c) a biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-15, and a pharmaceutically acceptable
excipient. In one embodiment, the pharmaceutical composition comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15. The invention additionally
provides a method of treating a disease or condition associated with decreased expression
of functional SYNT, comprising administering to a patient in need of such treatment
the pharmaceutical composition.
[0023] The invention also provides a method for screening a compound for effectiveness as
an agonist of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15, b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15,
c) a biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-15. The method comprises a) exposing
a sample comprising the polypeptide to a compound, and b) detecting agonist activity
in the sample. In one alternative, the invention provides a pharmaceutical composition
comprising an agonist compound identified by the method and a pharmaceutically acceptable
excipient. In another alternative, the invention provides a method of treating a disease
or condition associated with decreased expression of functional SYNT, comprising administering
to a patient in need of such treatment the pharmaceutical composition.
[0024] Additionally, the invention provides a method for screening a compound for effectiveness
as an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting of a) an amino acid sequence selected from the group consisting
of SEQ ID NO: 1-15, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-15, c) a biologically active fragment of an amino acid sequence selected
from the group consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of an
amino acid sequence selected from the group consisting of SEQ ID NO:1-15. The method
comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in the sample. In one alternative, the invention provides a pharmaceutical
composition comprising an antagonist compound identified by the method and a pharmaceutically
acceptable excipient. In another alternative, the invention provides a method of treating
a disease or condition associated with overexpression of functional SYNT, comprising
administering to a patient in need of such treatment the pharmaceutical composition.
[0025] The invention further provides a method of screening for a compound that specifically
binds to a polypeptide comprising an amino acid sequence selected from the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-15,
b) a naturally occurring amino acid sequence having at least 90% sequence identity
to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c)
a biologically active fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence selected
from the group consisting of SEQ ID NO:1-15. The method comprises a) combining the
polypeptide with at least one test compound under suitable conditions, and b) detecting
binding of the polypeptide to the test compound, thereby identifying a compound that
specifically binds to the polypeptide.
[0026] The invention further provides a method of screening for a compound that modulates
the activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an amino acid sequence selected from the group consisting of
SEQ ID NO:1-15, b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15,
c) a biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-15. The method comprises a) combining
the polypeptide with at least one test compound under conditions permissive for the
activity of the polypeptide, b) assessing the activity of the polypeptide in the presence
of the test compound, and c) comparing the activity of the polypeptide in the presence
of the test compound with the activity of the polypeptide in the absence of the test
compound, wherein a change in the activity of the polypeptide in the presence of the
test compound is indicative of a compound that modulates the activity of the polypeptide.
[0027] The invention further provides a method for screening a compound for effectiveness
in altering expression of a target polynucleotide, wherein said target polynucleotide
comprises a sequence selected from the group consisting of SEQ ID NO: 16-30, the method
comprising a) exposing a sample comprising the target polynucleotide to a compound,
and b) detecting altered expression of the target polynucleotide.
[0028] The invention further provides a method for assessing toxicity of a test compound,
said method comprising a) treating a biological sample containing nucleic acids with
the test compound; b) hybridizing the nucleic acids of the treated biological sample
with a probe comprising at least 20 contiguous nucleotides of a polynucleotide comprising
a polynucleotide sequence selected from the group consisting of i) a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 16-30. ii) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 16-30, iii) a polynucleotide
sequence complementary to i), iv) a polynucleotide sequence complementary to ii),
and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby
a specific hybridization complex is formed between said probe and a target polynucleotide
in the biological sample, said target polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:) 16-30, ii) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a polynucleotide
sequence selected from the group consisting of SEQ ID NO:16-30, iii) a polynucleotide
sequence complementary to i), iv) a polynucleotide sequence complementary to ii),
and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises
a fragment of the above polynucleotide sequence; c) quantifying the amount of hybridization
complex; and d) comparing the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated biological sample,
wherein a difference in the amount of hybridization complex in the treated biological
sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
[0029] Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID
NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-length sequences encoding SYNT.
[0030] Table 2 shows features of each polypeptide sequence, including potential motifs,
homologous sequences, and methods, algorithms, and searchable databases used for analysis
of SYNT.
[0031] Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific
expression patterns of each nucleic acid sequence as determined by northern analysis;
diseases. disorders, or conditions associated with these tissues; and the vector into
which each cDNA was cloned.
[0032] Table 4 describes the tissues used to construct the cDNA libraries from which cDNA
clones encoding SYNT were isolated.
[0033] Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides
and polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
[0034] Before the present proteins, nucleotide sequences, and methods are described, it
is understood that this invention is not limited to the particular machines. materials
and methods described, as these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention which will be limited only by
the appended claims.
[0035] It must be noted that as used herein and in the appended claims, the singular forms
"a," "an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a reference to "a host
-cell" includes a plurality of such host cells, and a reference to "an antibody" is
a reference to one or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0036] Unless defined otherwise, all technical and scientific terms used herein have the
same meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs. Although any machines, materials, and methods similar or equivalent
to those described herein can be used to practice or test the present invention, the
preferred machines, materials and methods are now described. All publications mentioned
herein are cited for the purpose of describing and disclosing the cell lines, protocols,
reagents and vectors which are reported in the publications and which might be used
in connection with the invention. Nothing herein is to be construed as an admission
that the invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
[0037] *'SYNT"' refers to the amino acid sequences of substantially purified SYNT obtained
from any species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic,
or recombinant.
[0038] The term "agonist" refers to a molecule which intensifies or mimics the biological
activity of SYNT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other compound or composition which modulates the activity of SYNT
either by directly interacting with SYNT or by acting on components of the biological
pathway in which SYNT participates.
[0039] An "allelic variant" is an alternative form of the gene encoding SYNT. Allelic variants
may result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in polypeptides whose structure or function may or may not be
altered. A gene may have none, one, or many allelic variants of its naturally occurring
form. Common mutational changes which give rise to allelic variants are generally
ascribed to natural deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the others, one or
more times in a given sequence.
[0040] "Altered" nucleic acid sequences encoding SYNT include those sequences with deletions,
insertions, or substitutions of different nucleotides, resulting in a polypeptide
the same as SYNT or a polypeptide with at least one functional characteristic of SYNT.
Included within this definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the polynucleotide encoding
SYNT, and improper or unexpected hybridization to allelic variants, with a locus other
than the normal chromosomal locus for the polynucleotide sequence encoding SYNT. The
encoded protein may also be "altered," and may contain deletions, insertions, or substitutions
of amino acid residues which produce a silent change and result in a functionally
equivalent SYNT. Deliberate amino acid substitutions may be made on the basis of similarity
in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues, as long as the biological or immunological activity of SYNT
is retained. For example, negatively charged amino acids may include aspartic acid
and glutamic acid, and positively charged amino acids may include lysine and arginine.
Amino acids with uncharged polar side chains having similar hydrophilicity values
may include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine,
and valine; glycine and alanine; and phenylalanine and tyrosine.
[0041] The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to naturally
occurring or synthetic molecules. Where "amino acid sequence" is recited to refer
to a sequence of a naturally occurring protein molecule, "amino acid sequence" and
like terms are not meant to limit the amino acid sequence to the complete native amino
acid sequence associated with the recited protein molecule.
[0042] "Amplification" relates to the production of additional copies of a nucleic acid
sequence. Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known in the art.
[0043] The term "antagonist" refers to a molecule which inhibits or attenuates the biological
activity of SYNT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small molecules, or any other compound or composition which modulates
the activity of SYNT either by directly interacting with SYNT or by acting on components
of the biological pathway in which SYNT participates.
[0044] The term "antibody" refers to intact immunoglobulin molecules as well as to fragments
thereof, such as Fab, F(ab')
2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies
that bind SYNT polypeptides can be prepared using intact polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be
derived from the translation of RNA, or synthesized chemically, and can be conjugated
to a carrier protein if desired. Commonly used carriers that are chemically coupled
to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0045] The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope)
that makes contact with a particular antibody. When a protein or a fragment of a protein
is used to immunize a host animal, numerous regions of the protein may induce the
production of antibodies which bind specifically to antigenic determinants (particular
regions or three-dimensional structures on the protein). An antigenic determinant
may compete with the intact antigen (i.e., the immunogen used to elicit the immune
response) for binding to an antibody.
[0046] The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include
DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars;
or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil,
or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including
chemical synthesis or transcription. Once introduced into a cell, the complementary
antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced
by the cell to form duplexes which block either transcription or translation. The
designation "negative" or "minus" can refer to the antisense strand, and the designation
"positive" or "plus" can refer to the sense strand of a reference DNA molecule.
[0047] The term "biologically active" refers to a protein having structural, regulatory,
or biochemical functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic" refers to the capability of the natural, recombinant, or
synthetic SYNT, or of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific antibodies.
[0048] "Complementary" describes the relationship between two single-stranded nucleic acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement,
3'-TCA-5'.
[0049] A "composition comprising a given polynucleotide sequence" and a "composition comprising
a given amino acid sequence" refer broadly to any composition containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide sequences encoding
SYNT or fragments of SYNT may be employed as hybridization probes. The probes may
be stored in freeze-dried form and may be associated with a stabilizing agent such
as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution
containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and
other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0050] "Consensus sequence" refers to a nucleic acid sequence which has been subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR
kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced,
or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA
fragments using a computer program for fragment assembly, such as the GELVIEW fragment
assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA).
Some sequences have been both extended and assembled to produce the consensus sequence.
[0051] "Conservative amino acid substitutions" are those substitutions that are predicted
to least interfere with the properties of the original protein, i.e., the structure
and especially the function of the protein is conserved and not significantly changed
by such substitutions. The table below shows amino acids which may be substituted
for an original amino acid in a protein and which are regarded as conservative amino
acid substitutions.
Original Residue |
Conservative Substitution |
Ala |
Gly, Ser |
Arg |
His, Lys |
Asn |
Asp, Gln, His |
Asp |
Asn, Glu |
Cys |
Ala, Ser |
Gln |
Asn, Glu, His |
Glu |
Asp, Gln, His |
Gly |
Ala |
His |
Asn, Arg, Gln, Glu |
Ile |
Leu, Val |
Leu |
Ile, Val |
Lys |
Arg, Gin, Glu |
Met |
Leu, Ile |
Phe |
His, Met, Leu, Trp, Tyr |
Ser |
Cys, Thr |
Thr |
Ser, Val |
Trp |
Phe, Tyr |
Tyr |
His, Phe, Trp |
Val |
Ile, Leu, Thr |
[0052] Conservative amino acid substitutions generally maintain (a) the structure of the
polypeptide backbone in the area of the substitution, for example, as a beta sheet
or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at
the site of the substitution, and/or (c) the bulk of the side chain.
[0053] A "deletion" refers to a change in the amino acid or nucleotide sequence that results
in the absence of one or more amino acid residues or nucleotides.
[0054] The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example, replacement
of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide
encodes a polypeptide which retains at least one biological or immunological function
of the natural molecule. A derivative polypeptide is one modified by glycosylation,
pegylation, or any similar process that retains at least one biological or immunological
function of the polypeptide from which it was derived.
[0055] A "detectable label" refers to a reporter molecule or enzyme that is capable of generating
a measurable signal and is covalently or noncovalently joined to a polynucleotide
or polypeptide.
[0056] A "fragment" is a unique portion of SYNT or the polynucleotide encoding SYNT which
is identical in sequence to but shorter in length than the parent sequence. A fragment
may comprise up to the entire length of the defined sequence, minus one nucleotide/amino
acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides
or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule,
or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75,
100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length.
Fragments may be preferentially selected from certain regions of a molecule. For example,
a polypeptide fragment may comprise a certain length of contiguous amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown
in a certain defined sequence. Clearly these lengths are exemplary, and any length
that is supported by the specification, including the Sequence Listing, tables, and
figures, may be encompassed by the present embodiments.
[0057] A fragment of SEQ ID NO:16-30 comprises a region of unique polynucleotide sequence
that specifically identifies SEQ ID NO:16-30, for example, as distinct from any other
sequence in the genome from which the fragment was obtained. A fragment of SEQ ID
NO:16-30 is useful, for example, in hybridization and amplification technologies and
in analogous methods that distinguish SEQ ID NO:16-30 from related polynucleotide
sequences. The precise length of a fragment of SEQ ID NO:16-30 and the region of SEQ
ID NO:16-30 to which the fragment corresponds are routinely determinable by one of
ordinary skill in the art based on the intended purpose for the fragment.
[0058] A fragment of SEQ ID NO:1-15 is encoded by a fragment of SEQ ID NO:16-30. A fragment
of SEQ ID NO:1-15 comprises a region of unique amino acid sequence that specifically
identifies SEQ ID NO:1-15. For example, a fragment of SEQ ID NO:1-15 is useful as
an immunogenic peptide for the development of antibodies that specifically recognize
SEQ ID NO:1-15. The precise length of a fragment of SEQ ID NO:1-15 and the region
of SEQ ID NO:1-15 to which the fragment corresponds are routinely determinable by
one of ordinary skill in the art based on the intended purpose for the fragment.
[0059] A "full-length" polynucleotide sequence is one containing at least a translation
initiation codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full-length" polynucleotide sequence encodes a "full-length"
polypeptide sequence.
[0060] "Homology" refers to sequence similarity or, interchangeably, sequence identity,
between two or more polynucleotide sequences or two or more polypeptide sequences.
[0061] The terms "percent identity" and "% identity," as applied to polynucleotide sequences,
refer to the percentage of residue matches between at least two polynucleotide sequences
aligned using a standardized algorithm. Such an algorithm may insert, in a standardized
and reproducible way, gaps in the sequences being compared in order to optimize alignment
between two sequences, and therefore achieve a more meaningful comparison of the two
sequences.
[0062] Percent identity between polynucleotide sequences may be determined using the default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment program. This program is part of the LASERGENE software package,
a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V
is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins.
D.G. et al. (1992) CABIOS 8: 189-19 1. For pairwise alignments of polynucleotide sequences,
the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and
"diagonals saved"=4. The "weighted" residue weight table is selected as the default.
Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned
polynucleotide sequences.
[0063] Alternatively, a suite of commonly used and freely available sequence comparison
algorithms is provided by the National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol.
215:403-4 10), which is available from several sources, including the NCBI, Bethesda.
MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software
suite includes various sequence analysis programs including "blastn," that is used
to align a known polynucleotide sequence with other polynucleotide sequences from
a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is
used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences"
can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/b12.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below).
BLAST programs are commonly used with gap and other parameters set to default settings.
For example, to compare two nucleotide sequences, one may use blastn with the "BLAST
2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default
parameters may be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 1
Filter: on
[0064] Percent identity may be measured over the length of an entire defined sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over the length of a fragment taken from a larger, defined sequence,
for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at
least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary
only, and it is understood that any fragment length supported by the sequences shown
herein, in the tables, figures, or Sequence Listing, may be used to describe a length
over which percentage identity may be measured.
[0065] Nucleic acid sequences that do not show a high degree of identity may nevertheless
encode similar amino acid sequences due to the degeneracy of the genetic code. It
is understood that changes in a nucleic acid sequence can be made using this degeneracy
to produce multiple nucleic acid sequences that all encode substantially the same
protein.
[0066] The phrases "percent identity" and "% identity," as applied to polypeptide sequences,
refer to the percentage of residue matches between at least two polypeptide sequences
aligned using a standardized algorithm. Methods of polypeptide sequence alignment
are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve
the charge and hydrophobicity at the site of substitution, thus preserving the structure
(and therefore function) of the polypeptide,
[0067] Percent identity between polypeptide sequences may be determined using the default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment program (described and referenced above). For pairwise alignments
of polypeptide sequences using CLUSTAL V, the default parameters are set as follows:
Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected
as the default residue weight table. As with polynucleotide alignments, the percent
identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide
sequence pairs.
[0068] Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool
Version 2.0.12 (Apr-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on
[0069] Percent identity may be measured over the length of an entire defined polypeptide
sequence, for example, as defined by a particular SEQ ID number, or may be measured
over a shorter length, for example, over the length of a fragment taken from a larger,
defined polypeptide sequence, for instance, a fragment of at least 15, at least 20,
at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
Such lengths are exemplary only, and it is understood that any fragment length supported
by the sequences shown herein, in the tables, figures or Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
[0070] "Human artificial chromosomes" (HACs) are linear microchromosomes which may contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements
required for chromosome replication, segregation and maintenance.
[0071] The term "humanized antibody" refers to an antibody molecule in which the amino acid
sequence in the non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its original binding ability.
[0072] "Hybridization" refers to the process by which a polynucleotide strand anneals with
a complementary strand through base pairing under defined hybridization conditions.
Specific hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity. Specific hybridization complexes form under permissive
annealing conditions and remain hybridized after the "washing" step(s). The washing
step(s) is particularly important in determining the stringency of the hybridization
process, with more stringent conditions allowing less non-specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable by one
of ordinary skill in the art and may be consistent among hybridization experiments.
whereas wash conditions may be varied among experiments to achieve the desired stringency,
and therefore hybridization specificity. Permissive annealing conditions occur, for
example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100
µg/ml sheared, denatured salmon sperm DNA.
[0073] Generally, stringency of hybridization is expressed, in part, with reference to the
temperature under which the wash step is carried out. Such wash temperatures are typically
selected to be about 5°C to 20°C lower than the thermal melting point (T
m) for the specific sequence at a defined ionic strength and pH. The T
m is the temperature (under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. An equation for calculating T
m and conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al., 1989,
Molecular Cloning: A Laboratory Manual, 2
nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2,
chapter 9.
[0074] High stringency conditions for hybridization between polynucleotides of the present
invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about
0.1% SDS. for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C
may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being
present at about 0.1%. Typically, blocking reagents are used to block non-specific
hybridization. Such blocking reagents include, for instance, sheared and denatured
salmon sperm DNA at about 100-200 µg/ml. Organic solvent, such as formamide at a concentration
of about 35-50% v/v, may also be used under particular circumstances, such as for
RNA:DNA hybridizations. Useful variations on these wash conditions will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly under
high stringency conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides
and their encoded polypeptides.
[0075] The term "hybridization complex" refers to a complex formed between two nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary bases.
A hybridization complex may be formed in solution (e.g., C
0t or R
0t analysis) or formed between one nucleic acid sequence present in solution and another
nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters,
chips, pins or glass slides, or any other appropriate substrate to which cells or
their nucleic acids have been fixed).
[0076] The words "-insertion" and "addition" refer to changes in an amino acid or nucleotide
sequence resulting in the addition of one or more amino acid residues or nucleotides,
respectively.
[0077] "Immune response" can refer to conditions associated with inflammation, trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be characterized
by expression of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect cellular and systemic defense systems.
[0078] An "immunogenic fragment" is a polypeptide or oligopeptide fragment of SYNT which
is capable of eliciting an immune response when introduced into a living organism,
for example, a mammal. The term "immunogenic fragment" also includes any polypeptide
or oligopeptide fragment of SYNT which is useful in any of the antibody production
methods disclosed herein or known in the art.
[0079] The term "microarray" refers to an arrangement of a plurality of polynucleotides,
polypeptides, or other chemical compounds on a substrate.
[0080] The terms "element" and "array element" refer to a polynucleotide, polypeptide, or
other chemical compound having a unique and defined position on a microarray.
[0081] The term "modulate" refers to a change in the activity of SYNT. For example, modulation
may cause an increase or a decrease in protein activity, binding characteristics,
or any other biological, functional, or immunological properties of SYNT.
[0082] The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of
genomic or synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to
any DNA-like or RNA-like material.
[0083] "Operably linked" refers to the situation in which a first nucleic acid sequence
is placed in a functional relationship with a second nucleic acid sequence. For instance,
a promoter is operably linked to a coding sequence if the promoter affects the transcription
or expression of the coding sequence. Operably linked DNA sequences may be in close
proximity or contiguous and, where necessary to join two protein coding regions, in
the same reading frame.
[0084] "Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked to a
peptide backbone of amino acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind complementary single stranded
DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan
in the cell.
[0085] "Post-translational modification" of an SYNT may involve lipidation, glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications
known in the art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by cell type depending on the enzymatic milieu of SYNT.
[0086] "Probe" refers to nucleic acid sequences encoding SYNT, their complements, or fragments
thereof, which are used to detect identical, allelic or related nucleic acid sequences.
Probes are isolated oligonucleotides or polynucleotides attached to a detectable label
or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent
agents, and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides,
which may be annealed to a target polynucleotide by complementary base-pairing. The
primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
Primer pairs can be used for amplification (and identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
[0087] Probes and primers as used in the present invention typically comprise at least 15
contiguous nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also be employed, such as probes and primers that comprise
at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides
of the disclosed nucleic acid sequences. Probes and primers may be considerably longer
than these examples, and it is understood that any length supported by the specification,
including the tables, figures, and Sequence Listing, may be used.
[0088] Methods for preparing and using probes and primers are described in the references,
for example Sambrook, J. et al., 1989,
Molecular Cloning: A Laboratory Manual, 2
nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al.,1987,
Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al., 1990,
PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence,
for example, by using computer programs intended for that purpose such as Primer (Version
0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
[0089] Oligonucleotides for use as primers are selected using software known in the art
for such purpose. For example, OLIGO 4.06 software is useful for the selection of
PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides
and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide
sequence of up to 32 kilobases. Similar primer selection programs have incorporated
additional features for expanded capabilities. For example, the PrimOU primer selection
program (available to the public from the Genome Center at University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection program (available to the public from the Whitehead Institute/MIT
Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library,"
in which sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the selection of oligonucleotides for microarrays. (The
source code for the latter two primer selection programs may also be obtained from
their respective sources and modified to meet the user's specific needs.) The PrimeGen
program (available to the public from the UK Human Genome Mapping Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby
allowing selection of primers that hybridize to either the most conserved or least
conserved regions of aligned nucleic acid sequences. Hence, this program is useful
for identification of both unique and conserved oligonucleotides and polynucleotide
fragments. The oligonucleotides and polynucleotide fragments identified by any of
the above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to identify
fully or partially complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide selection are not limited to those described above.
[0090] A "recombinant nucleic acid" is a sequence that is not naturally occurring or has
a sequence that is made by an artificial combination of two or more otherwise separated
segments of sequence. This artificial combination is often accomplished by chemical
synthesis or, more commonly, by the artificial manipulation of isolated segments of
nucleic acids, e.g., by genetic engineering techniques such as those described in
Sambrook,
supra. The term recombinant includes nucleic acids that have been altered solely by addition,
substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant
nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example,
to transform a cell.
[0091] Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g.,
based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant
nucleic acid is expressed, inducing a protective immunological response in the mammal.
[0092] A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated
regions (UTRs). Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA stability.
[0093] "Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic
acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
[0094] An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear
sequence of nucleotides as the reference DNA sequence with the exception that all
occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar
backbone is composed of ribose instead of deoxyribose.
[0095] The term "sample" is used in its broadest sense. A sample suspected of containing
nucleic acids encoding SYNT, or fragments thereof, or SYNT itself, may comprise a
bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated
from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate;
a tissue; a tissue print; etc.
[0096] The terms "specific binding" and "specifically binding" refer to that interaction
between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule,
or any natural or synthetic binding composition. The interaction is dependent upon
the presence of a particular structure of the protein, e.g., the antigenic determinant
or epitope, recognized by the binding molecule. For example, if an antibody is specific
for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence
of free unlabeled A, in a reaction containing free labeled A and the antibody will
reduce the amount of labeled A that binds to the antibody.
[0097] The term "substantially purified" refers to nucleic acid or amino acid sequences
that are removed from their natural environment and are isolated or separated, and
are at least 60% free, preferably at least 75% free, and most preferably at least
90% free from other components with which they are naturally associated.
[0098] A "substitution" refers to the replacement of one or more amino acid residues or
nucleotides by different amino acid residues or nucleotides, respectively.
[0099] "Substrate" refers to any suitable rigid or semi-rigid support including membranes,
filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers, microparticles and capillaries. The substrate can have a variety
of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides
or polypeptides are bound.
[0100] A "transcript image" refers to the collective pattern of gene expression by a particular
cell type or tissue under given conditions at a given time.
[0101] "Transformation" describes a process by which exogenous DNA is introduced into a
recipient cell. Transformation may occur under natural or artificial conditions according
to various methods well known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host
cell. The method for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term "transformed"
cells includes stably transformed cells in which the inserted DNA is capable of replication
either as an autonomously replicating plasmid or as part of the host chromosome, as
well as transiently transformed cells which express the inserted DNA or RNA for limited
periods of time.
[0102] A "transgenic organism," as used herein, is any organism, including but not limited
to animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid introduced by way of human intervention, such as by transgenic
techniques well known in the art. The nucleic acid is introduced into the cell, directly
or indirectly by introduction into a precursor of the cell, by way of deliberate genetic
manipulation, such as by microinjection or by infection with a recombinant virus.
The term genetic manipulation does not include classical cross-breeding, or
in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
The transgenic organisms contemplated in accordance with the present invention include
bacteria, cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be introduced into the host by methods known in the art, for example
infection, transfection, transformation or transconjugation. Techniques for transferring
the DNA of the present invention into such organisms are widely known and provided
in references such as Sambrook et al. (1989),
supra.
[0103] A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence
having at least 40% sequence identity to the particular nucleic acid sequence over
a certain length of one of the nucleic acid sequences using blastn with the "BLAST
2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters. Such a pair
of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%,
at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater
sequence identity over a certain defined length. A variant may be described as, for
example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant.
A splice variant may have significant identity to a reference molecule, but will generally
have a greater or lesser number of polynucleotides due to alternative splicing of
exons during mRNA processing. The corresponding polypeptide may possess additional
functional domains or lack domains that are present in the reference molecule. Species
variants are polynucleotide sequences that vary from one species to another. The resulting
polypeptides generally will have significant amino acid identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of a particular
gene between individuals of a given species. Polymorphic variants also may encompass
"single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies
by one nucleotide base. The presence of SNPs may be indicative of, for example, a
certain population, a disease state, or a propensity for a disease state.
[0104] A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence
having at least 40% sequence identity to the particular polypeptide sequence over
a certain length of one of the polypeptide sequences using blastp with the "BLAST
2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters. Such a pair
of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity
over a certain defined length of one of the polypeptides.
THE INVENTION
[0105] The invention is based on the discovery of new human synthetases (SYNT), the polynucleotides
encoding SYNT, and the use of these compositions for the diagnosis, treatment, or
prevention of immune, neuronal, and reproductive disorders, and cell proliferative
disorders including cancer.
[0106] Table 1 lists the Incyte clones used to assemble full length nucleotide sequences
encoding SYNT. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs)
of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte clones in which nucleic acids encoding each SYNT were identified.
and column 4 shows the cDNA libraries from which these clones were isolated. Column
5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA
libraries are not indicated were derived from pooled cDNA libraries. In some cases,
GenBank sequence identifiers are also shown in column 5. The Incyte clones and GenBank
cDNA sequences, where indicated, in column 5 were used to assemble the consensus nucleotide
sequence of each SYNT and are useful as fragments in hybridization technologies.
[0107] The columns of Table 2 show various properties of each of the polypeptides of the
invention: column 1 references the SEQ ID NO: column 2 shows the number of amino acid
residues in each polypeptide; column 3 shows potential phosphorylation sites; column
4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising
signature sequences and motifs; column 6 shows homologous sequences as identified
by BLAST analysis; and column 7 shows analytical methods and in some cases, searchable
databases to which the analytical methods were applied. The methods of column 7 were
used to characterize each polypeptide through sequence homology and protein motifs.
[0108] The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions
associated with nucleotide sequences encoding SYNT. The first column of Table 3 lists
the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of
column 1. These fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22. SEQ ID NO:23, SEQ ID NO:25. SEQ ID NO:26, SEQ
ID NO:27, SEQ ID NO:29, and SEQ ID NO:30 and to distinguish between SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 SEQ ID NO:20. SEQ ID NO:21, SEQ ID NO:22.
SEQ ID NO:23. SEQ ID NO:25, SEQ ID NO:26. SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:30
and related polynucleotide sequences. The polypeptides encoded by these fragments
are useful, for example, as immunogenic peptides. Column 3 lists tissue categories
which express SYNT as a fraction of total tissues expressing SYNT. Column 4 lists
diseases, disorders, or conditions associated with those tissues expressing SYNT as
a fraction of total tissues expressing SYNT. Column 5 lists the vectors used to subclone
each cDNA library.
[0109] The columns of Table 4 show descriptions of the tissues used to construct the cDNA
libraries from which cDNA clones encoding SYNT were isolated. Column 1 references
the nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these clones
were isolated, and column 3 shows the tissue origins and other descriptive information
relevant to the cDNA libraries in column 2.
[0110] SEQ ID NO: 16 maps to chromosome 5 within the interval from 147.10 to 150.00 centiMorgans.
SEQ ID NO: 17 maps to chromosome 10 within the interval from 137.60 to 139.20 centiMorgans.
This interval also contains gene MXI1, a member of the MYC family. SEQ ID NO: 18 maps
to chromosome 2 within the interval from 228.80 to 230.10 centiMorgans. This interval
also contains a gene for a proto-oncogene encoding a tyrosine-protein kinase. SEQ
ID NO:21 maps to chromosome 5 within the interval from 172.6 to 184.7 centiMorgans.
SEQ ID NO:24 maps to chromosome 2 within the interval from 118.0 to 127.4 centiMorgans.
SEQ ID NO:26 maps to chromosome 3 within the interval from 157.4 to 162.0 centiMorgans.
SEQ ID NO:27 maps to chromosome 12 within the interval from 97.1 to 116.6 centiMorgans.
SEQ ID NO:28 maps to chromosome 4 within the interval from 77.3 to 99.2 centiMorgans
and to chromosome 5 within the intervals from 79.2 to 92.3 centiMorgans, from 116.3
to 127.9 centiMorgans, and from 157.6 to 163.0 centiMorgans. SEQ ID NO:29 maps to
chromosome 1 within the interval from 242.5 to 258.7 centiMorgans and to chromosome
19 within the interval from 69.9 to 104.9 centiMorgans. SEQ ID NO:30 maps to chromosome
1 within the interval from 57.2 to 57.5 centiMorgans.
[0111] The invention also encompasses SYNT variants. A preferred SYNT variant is one which
has at least about 80%, or alternatively at least about 90%, or even at least about
95% amino acid sequence identity to the SYNT amino acid sequence, and which contains
at least one functional or structural characteristic of SYNT.
[0112] The invention also encompasses polynucleotides which encode SYNT. In a particular
embodiment, the invention encompasses a polynucleotide sequence comprising a sequence
selected from the group consisting of SEQ ID NO:16-30, which encodes SYNT. The polynucleotide
sequences of SEQ ID NO:16-30, as presented in the Sequence Listing, embrace the equivalent
RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with
uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
[0113] The invention also encompasses a variant of a polynucleotide sequence encoding SYNT.
In particular, such a variant polynucleotide sequence will have at least about 70%.
or alternatively at least about 85%, or even at least about 95% polynucleotide sequence
identity to the polynucleotide sequence encoding SYNT. A particular aspect of the
invention encompasses a variant of a polynucleotide sequence comprising a sequence
selected from the group consisting of SEQ ID NO:16-30 which has at least about 70%,
or alternatively at least about 85%, or even at least about 95% polynucleotide sequence
identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:16-30.
Any one of the polynucleotide variants described above can encode an amino acid sequence
which contains at least one functional or structural characteristic of SYNT.
[0114] It will be appreciated by those skilled in the art that as a result of the degeneracy
of the genetic code, a multitude of polynucleotide sequences encoding SYNT, some bearing
minimal similarity to the polynucleotide sequences of any known and naturally occurring
gene, may be produced. Thus, the invention contemplates each and every possible variation
of polynucleotide sequence that could be made by selecting combinations based on possible
codon choices. These combinations are made in accordance with the standard triplet
genetic code as applied to the polynucleotide sequence of naturally occurring SYNT,
and all such variations are to be considered as being specifically disclosed.
[0115] Although nucleotide sequences which encode SYNT and its variants are generally capable
of hybridizing to the nucleotide sequence of the naturally occurring SYNT under appropriately
selected conditions of stringency, it may be advantageous to produce nucleotide sequences
encoding SYNT or its derivatives possessing a substantially different codon usage.
e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase
the rate at which expression of the peptide occurs in a particular prokaryotic or
eukaryotic host in accordance with the frequency with which particular codons are
utilized by the host. Other reasons for substantially altering the nucleotide sequence
encoding SYNT and its derivatives without altering the encoded amino acid sequences
include the production of RNA transcripts having more desirable properties, such as
a greater half-life, than transcripts produced from the naturally occurring sequence.
[0116] The invention also encompasses production of DNA sequences which encode SYNT and
SYNT derivatives, or fragments thereof, entirely by synthetic chemistry. After production,
the synthetic sequence may be inserted into any of the many available expression vectors
and cell systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to introduce mutations into a sequence encoding SYNT or any fragment thereof.
[0117] Also encompassed by the invention are polynucleotide sequences that are capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to those
shown in SEQ ID NO:16-30 and fragments thereof under various conditions of stringency.
(See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel,
A.R. (1987) Methods Enzymol. 152:507-511.) Hybridization conditions, including annealing
and wash conditions, are described in "Definitions."
[0118] Methods for DNA sequencing are well known in the art and may be used to practice
any of the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of DNA polymerase I. SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerase (PE Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia
Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases
such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg
MD). Preferably, sequence preparation is automated with machines such as the MICROLAB
2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research,
Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then
carried out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems),
the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other
systems known in the art. The resulting sequences are analyzed using a variety of
algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997)
Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7: Meyers, R.A. (1995)
Molecular Biology and Biotechnology, Wiley VCH. New York NY, pp. 856-853.)
[0119] The nucleic acid sequences encoding SYNT may be extended utilizing a partial nucleotide
sequence and employing various PCR-based methods known in the art to detect upstream
sequences, such as promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal and nested primers to amplify
unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G.
(1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that
extend in divergent directions to amplify unknown sequence from a circularized template.
The template is derived from restriction fragments comprising a known genomic locus
and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments
adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert an engineered double-stranded
sequence into a region of unknown sequence before performing PCR. Other methods which
may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker.
J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,
nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic
DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon
junctions. For all PCR-based methods, primers may be designed using commercially available
software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth
MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°C to 72°C.
[0120] When screening for full-length cDNAs, it is preferable to use libraries that have
been size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include sequences containing the 5' regions of genes, are preferable for
situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic
libraries may be useful for extension of sequence into 5' non-transcribed regulatory
regions.
[0121] Capillary electrophoresis systems which are commercially available may be used to
analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
In particular, capillary sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated fluorescent dyes,
and a charge coupled device camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate software (e.g.,
GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the entire process from loading
of samples to computer analysis and electronic data display may be computer controlled.
Capillary electrophoresis is especially preferable for sequencing small DNA fragments
which may be present in limited amounts in a particular sample.
[0122] In another embodiment of the invention, polynucleotide sequences or fragments thereof
which encode SYNT may be cloned in recombinant DNA molecules that direct expression
of SYNT, or fragments or functional equivalents thereof, in appropriate host cells.
Due to the inherent degeneracy of the genetic code, other DNA sequences which encode
substantially the same or a functionally equivalent amino acid sequence may be produced
and used to express SYNT.
[0123] The nucleotide sequences of the present invention can be engineered using methods
generally known in the art in order to alter SYNT-encoding sequences for a variety
of purposes including, but not limited to, modification of the cloning, processing,
and/or expression of the gene product. DNA shuffling by random fragmentation and PCR
reassembly of gene fragments and synthetic oligonucleotides may be used to engineer
the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis
may be used to introduce mutations that create new restriction sites, alter glycosylation
patterns, change codon preference, produce splice variants, and so forth.
[0124] The nucleotides of the present invention may be subjected to DNA shuffling techniques
such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA: described in U.S. Patent
Number 5.837.458: Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797: Christians.
F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri. A. et al. ( 1996) Nat.
Biotechnol. 14:315-319) to alter or improve the biological properties of SYNT, such
as its biological or enzymatic activity or its ability to bind to other molecules
or compounds. DNA shuffling is a process by which a library of gene variants is produced
using PCR-mediated recombination of gene fragments. The library is then subjected
to selection or screening procedures that identify those gene variants with the desired
properties. These preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial" breeding and rapid molecular evolution. For example, fragments
of a single gene containing random point mutations may be recombined, screened, and
then reshuffled until the desired properties are optimized. Alternatively, fragments
of a given gene may be recombined with fragments of homologous genes in the same gene
family, either from the same or different species, thereby maximizing the genetic
diversity of multiple naturally occurring genes in a directed and controllable manner.
[0125] In another embodiment, sequences encoding SYNT may be synthesized, in whole or in
part, using chemical methods well known in the art. (See, e.g., Caruthers. M.H. et
al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn. T. et al. (1980) Nucleic
Acids Symp. Ser. 7:225-232.) Alternatively. SYNT itself or a fragment thereof may
be synthesized using chemical methods. For example, peptide synthesis can be performed
using various solution-phase or solid-phase techniques. (See, e.g., Creighton. T.
(1984)
Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60: and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis may be achieved using the ABI 431A peptide synthesizer (PE Biosystems).
Additionally, the amino acid sequence of SYNT, or any part thereof, may be altered
during direct synthesis and/or combined with sequences from other proteins, or any
part thereof, to produce a variant polypeptide or a polypeptide having a sequence
of a naturally occurring polypeptide.
[0126] The peptide may be substantially purified by preparative high performance liquid
chromatography. (See, e.g., Chiez. R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton,
supra, pp. 28-53.)
[0127] In order to express a biologically active SYNT, the nucleotide sequences encoding
SYNT or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains the necessary elements for transcriptional and translational
control of the inserted coding sequence in a suitable host. These elements include
regulatory sequences, such as enhancers, constitutive and inducible promoters, and
5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding
SYNT. Such elements may vary in their strength and specificity. Specific initiation
signals may also be used to achieve more efficient translation of sequences encoding
SYNT. Such signals include the ATG initiation codon and adjacent sequences, e.g. the
Kozak sequence. In cases where sequences encoding SYNT and its initiation codon and
upstream regulatory sequences are inserted into the appropriate expression vector,
no additional transcriptional or translational control signals may be needed. However,
in cases where only coding sequence, or a fragment thereof, is inserted, exogenous
translational control signals including an in-frame ATG initiation codon should be
provided by the vector. Exogenous translational elements and initiation codons may
be of various origins, both natural and synthetic. The efficiency of expression may
be enhanced by the inclusion of enhancers appropriate for the particular host cell
system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
[0128] Methods which are well known to those skilled in the art may be used to construct
expression vectors containing sequences encoding SYNT and appropriate transcriptional
and translational control elements. These methods include
in vitro recombinant DNA techniques, synthetic techniques, and
in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al.
(1995)
Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9, 13, and 16.)
[0129] A variety of expression vector/host systems may be utilized to contain and express
sequences encoding SYNT. These include, but are not limited to, microorganisms such
as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect cell systems infected
with viral expression vectors (e.g., baculovirus); plant cell systems transformed
with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook.
supra; Ausubel,
supra: Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509-. Bitter. G.A.
et al. (1987) Methods Enzymol. 153:516-544; Scorer. C.A. et al. (1994) Bio/Technology
12:181-184; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227;
Sandig. V. et al. (1996) Hum. Gene Ther. 7:1937-1945. Takamatsu. N. (1987) EMBO J.
6:307-311; Coruzzi. G. et al. (1984) EMBO J. 3:1671-1680: Broglie, R. et al. (1984)
Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105:
The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill. New York NY,
pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659:
and Harrington. J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived
from retroviruses, adenoviruses, or herpes or vaccinia viruses. or from various bacterial
plasmids, may be used for delivery of nucleotide sequences to the targeted organ,
tissue, or cell population. (See, e.g.. Di Nicola, M. et al. (1998) Cancer Gen. Ther.
5(6):350-356; Yu, M. et al., (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344: Buller,
R.M. et al. (1985) Nature 317(6040):813-815; McGregor. D.P. et al. (1994) Mol. Immunol.
31(3):219-226; and Verma, I.M. and N. Somia (1997) Nature 389:239-242.) The invention
is not limited by the host cell employed.
[0130] In bacterial systems, a number of cloning and expression vectors may be selected
depending upon the use intended for polynucleotide sequences encoding SYNT. For example,
routine cloning, subcloning, and propagation of polynucleotide sequences encoding
SYNT can be achieved using a multifunctional
E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies).
Ligation of sequences encoding SYNT into the vector's multiple cloning site disrupts
the
lacZ gene, allowing a colorimetric screening procedure for identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be useful
for
in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation
of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509.) When large quantities of SYNT are needed, e.g.
for the production of antibodies, vectors which direct high level expression of SYNT
may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage
promoter may be used.
[0131] Yeast expression systems may be used for production of SYNT. A number of vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase,
and PGH promoters, may be used in the yeast
Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention
of expressed proteins and enable integration of foreign sequences into the host genome
for stable propagation. (See, e.g., Ausubel, 1995,
supra; Bitter,
supra; and Scorer,
supra.)
[0132] Plant systems may also be used for expression of SYNT. Transcription of sequences
encoding SYNT may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit
of RUBISCO or heat shock promoters may be used. (See. e.g., Coruzzi,
supra: Broglie.
supra: and Winter,
supra.) These constructs can be introduced into plant cells by direct DNA transformation
or pathogen-mediated transfection. (See, e.g.,
The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.)
[0133] In mammalian cells, a number of viral-based expression systems may be utilized. In
cases where an adenovirus is used as an expression vector, sequences encoding SYNT
may be ligated into an adenovirus transcription/translation complex consisting of
the late promoter and tripartite leader sequence. Insertion in a non-essential E1
or E3 region of the viral genome may be used to obtain infective virus which expresses
SYNT in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
[0134] Human artificial chromosomes (HACs) may also be employed to deliver larger fragments
of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to
10.Mb are constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington,
JJ. et al. (1997) Nat. Genet. 15:345-355.)
[0135] For long term production of recombinant proteins in mammalian systems, stable expression
of SYNT in cell lines is preferred. For example, sequences encoding SYNT can be transformed
into cell lines using expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene on the same or
on a separate vector. Following the introduction of the vector, cells may be allowed
to grow for about 1 to 2 days in enriched media before being switched to selective
media. The purpose of the selectable marker is to confer resistance to a selective
agent, and its presence allows growth and recovery of cells which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be propagated
using tissue culture techniques appropriate to the cell type.
[0136] Any number of selection systems may be used to recover transformed cell lines. These
include, but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase genes. for use in
tk- and
apr- cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy,
I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide
resistance can be used as the basis for selection. For example,
dhfr confers resistance to methotrexate;
neo confers resistance to the aminoglycosides neomycin and G-418; and
als and
pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin,
F. et al, (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described.
e.g..
trpB and
hisD, which alter cellular requirements for metabolites. (See. e.g., Hartman. S.C. and
R.C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g.,
anthocyanins, green fluorescent proteins (GFP: Clontech), β glucuronidase and its
substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These
markers can be used not only to identify transformants, but also to quantify the amount
of transient or stable protein expression attributable to a specific vector system.
(See. e.g., Rhodes. C.A. (1995) Methods Mol. Biol. 55:121-131.)
[0137] Although the presence/absence of marker gene expression suggests that the gene of
interest is also present, the presence and expression of the gene may need to be confirmed.
For example, if the sequence encoding SYNT is inserted within a marker gene sequence,
transformed cells containing sequences encoding SYNT can be identified by the absence
of marker gene function. Alternatively, a marker gene can be placed in tandem with
a sequence encoding SYNT under the control of a single promoter. Expression of the
marker gene in response to induction or selection usually indicates expression of
the tandem gene as well.
[0138] In general, host cells that contain the nucleic acid sequence encoding SYNT and that
express SYNT may be identified by a variety of procedures known to those of skill
in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR amplification, and protein bioassay or immunoassay techniques which include membrane,
solution, or chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
[0139] Immunological methods for detecting and measuring the expression of SYNT using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of such
techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays
(RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes
on SYNT is preferred, but a competitive binding assay may be employed. These and other
assays are well known in the art. (See, e.g., Hampton, R. et al. (1990)
Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997)
Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998)
Immunochemical Protocols, Humana Press. Totowa NJ.)
[0140] A wide variety of labels and conjugation techniques are known by those skilled in
the art and may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization or PCR probes for detecting sequences related to polynucleotides
encoding SYNT include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide. Alternatively, the sequences encoding SYNT, or any fragments
thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors
are known in the art, are commercially available, and may be used to synthesize RNA
probes
in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures may be conducted using a variety of commercially available kits,
such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US
Biochemical. Suitable reporter molecules or labels which may be used for ease of detection
include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents,
as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
[0141] Host cells transformed with nucleotide sequences encoding SYNT may be cultured under
conditions suitable for the expression and recovery of the protein from cell culture.
The protein produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence and/or the vector used. As will be understood by those of
skill in the art, expression vectors containing polynucleotides which encode SYNT
may be designed to contain signal sequences which direct secretion of SYNT through
a prokaryotic or eukaryotic cell membrane.
[0142] In addition, a host cell strain may be chosen for its ability to modulate expression
of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational
processing which cleaves a "prepro" or "pro" form of the protein may also be used
to specify protein targeting, folding, and/or activity. Different host cells which
have specific cellular machinery and characteristic mechanisms for post-translational
activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American
Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and processing of the foreign protein.
[0143] In another embodiment of the invention, natural, modified, or recombinant nucleic
acid sequences encoding SYNT may be ligated to a heterologous sequence resulting in
translation of a fusion protein in any of the aforementioned host systems. For example,
a chimeric SYNT protein containing a heterologous moiety that can be recognized by
a commercially available antibody may facilitate the screening of peptide libraries
for inhibitors of SYNT activity. Heterologous protein and peptide moieties may also
facilitate purification of fusion proteins using commercially available affinity matrices.
Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His,
FLAG,
c-myc, and hemagglutinin (HA), GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins, respectively. FLAG,
c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a proteolytic
cleavage site located between the SYNT encoding sequence and the heterologous protein
sequence, so that SYNT may be cleaved away from the heterologous moiety following
purification. Methods for fusion protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10). A variety of commercially available kits may also be used to facilitate
expression and purification of fusion proteins.
[0144] In a further embodiment of the invention, synthesis of radiolabeled SYNT may be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega).
These systems couple transcription and translation of protein-coding sequences operably
associated with the T7, T3, or SP6 promoters. Translation takes place in the presence
of a radiolabeled amino acid precursor, for example,
35S-methionine.
[0145] SYNT of the present invention or fragments thereof may be used to screen for compounds
that specifically bind to SYNT. At least one and up to a plurality of test compounds
may be screened for specific binding to SYNT. Examples of test compounds include antibodies,
oligonucleotides, proteins (e.g., receptors), or small molecules.
[0146] In one embodiment, the compound thus identified is closely related to the natural
ligand of SYNT, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a natural binding partner. (See, Coligan, J.E. et al. (1991)
Current Protocols in Immunology 1(2): Chapter 5.) Similarly, the compound can be closely related to the natural receptor
to which SYNT binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the compound can be rationally designed using known techniques.
In one embodiment, screening for these compounds involves producing appropriate cells
which express SYNT, either as a secreted protein or on the cell membrane. Preferred
cells include cells from mammals, yeast,
Drosophila, or
E. coli. Cells expressing SYNT or cell membrane fractions which contain SYNT are then contacted
with a test compound and binding, stimulation, or inhibition of activity of either
SYNT or the compound is analyzed.
[0147] An assay may simply test binding of a test compound to the polypeptide, wherein binding
is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the assay may comprise the steps of combining at least one test
compound with SYNT, either in solution or affixed to a solid support, and detecting
the binding of SYNT to the compound. Alternatively, the assay may detect or measure
binding of a test compound in the presence of a labeled competitor. Additionally,
the assay may be carried out using cell-free preparations, chemical libraries, or
natural product mixtures, and the test compound(s) may be free in solution or affixed
to a solid support.
[0148] SYNT of the present invention or fragments thereof may be used to screen for compounds
that modulate the activity of SYNT. Such compounds may include agonists, antagonists,
or partial or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for SYNT activity, wherein SYNT is combined with at least one test compound,
and the activity of SYNT in the presence of a test compound is compared with the activity
of SYNT in the absence of the test compound. A change in the activity of SYNT in the
presence of the test compound is indicative of a compound that modulates the activity
of SYNT. Alternatively, a test compound is combined with an
in vitro or cell-free system comprising SYNT under conditions suitable for SYNT activity,
and the assay is performed. In either of these assays, a test compound which modulates
the activity of SYNT may do so indirectly and need not come in direct contact with
the test compound. At least one and up to a plurality of test compounds may be screened.
[0149] In another embodiment, polynucleotides encoding SYNT or their mammalian homologs
may be "knocked out" in an animal model system using homologous recombination in embryonic
stem (ES) cells. Such techniques are well known in the art and are useful for the
generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383
and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ
cell line, are derived from the early mouse embryo and grown in culture. The ES cells
are transformed with a vector containing the gene of interest disrupted by a marker
gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science
244:1288-1292). The vector integrates into the corresponding region of the host genome
by homologous recombination. Alternatively, homologous recombination takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002: Wagner, K.U. et al. (1997)
Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected
into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts
are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny
are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals
thus generated may be tested with potential therapeutic or toxic agents.
[0150] Polynucleotides encoding SYNT may also be manipulated
in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to
differentiate into at least eight separate cell lineages including endoderm, mesoderm,
and ectodermal cell types. These cell lineages differentiate into, for example, neural
cells, hematopoietic lineages, and cardiomyocytes (Thomson. J.A. et al. (1998) Science
282:1145-1147).
[0151] Polynucleotides encoding SYNT can also be used to create "knockin" humanized animals
(pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology,
a region of a polynucleotide encoding SYNT is injected into animal ES cells, and the
injected sequence integrates into the animal cell genome. Transformed cells are injected
into blastulae, and the blastulae are implanted as described above. Transgenic progeny
or inbred lines are studied and treated with potential pharmaceutical agents to obtain
information on treatment of a human disease. Alternatively, a mammal inbred to overexpress
SYNT, e.g., by secreting SYNT in its milk, may also serve as a convenient source of
that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
[0152] Chemical and structural similarity, e.g., in the context of sequences and motifs,
exists between regions of SYNT and human synthetases. In addition, the expression
of SYNT is closely associated with hematopoietic/immune, cancerous, proliferating,
inflamed, immune, nervous, gastrointestinal and reproductive tissues. Therefore, SYNT
appears to play a role in an immune disorder such as inflammation, actinic keratosis,
acquired immunodeficiency syndrome (AIDS). Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis.
hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins,
mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial
or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis,
polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,
scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, trauma, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and hematopoietic cancer including lymphoma, leukemia, and myeloma; a
neuronal disorder, such as akathesia, Alzheimer's disease, amnesia, amyotrophic lateral
sclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia, depression,
diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy, Huntington's
disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's
disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's
disorder; a reproductive disorder, such as a disorder of prolactin production, infertility,
including tubal disease, ovulatory defects, and endometriosis, a disruption of the
estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune
disorders, an ectopic pregnancy, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia,
prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
and a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia,
and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
[0153] In another embodiment. a vector capable of expressing SYNT or a fragment or derivative
thereof may be administered to a subject to treat or prevent a disorder associated
with decreased expression or activity of SYNT including, but not limited to, those
described above.
[0154] In a further embodiment, a pharmaceutical composition comprising a substantially
purified SYNT in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat or prevent a disorder associated with decreased expression or
activity of SYNT including, but not limited to, those provided above.
[0155] In still another embodiment, an agonist which modulates the activity of SYNT may
be administered to a subject to treat or prevent a disorder associated with decreased
expression or activity of SYNT including, but not limited to, those listed above.
[0156] In a further embodiment, an antagonist of SYNT may be administered to a subject to
treat or prevent a disorder associated with increased expression or activity of SYNT.
Examples of such disorders include, but are not limited to, those immune, neuronal,
reproductive, and cell proliferative disorders described above. In one aspect, an
antibody which specifically binds SYNT may be used directly as an antagonist or indirectly
as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express SYNT.
[0157] In an additional embodiment, a vector expressing the complement of the polynucleotide
encoding SYNT may be administered to a subject to treat or prevent a disorder associated
with increased expression or activity of SYNT including, but not limited to, those
described above.
[0158] In other embodiments, any of the proteins, antagonists, antibodies. agonists, complementary
sequences, or vectors of the invention may be administered in combination with other
appropriate therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made by one of ordinary skill in the art, according to conventional
pharmaceutical principles. The combination of therapeutic agents may act synergistically
to effect the treatment or prevention of the various disorders described above. Using
this approach, one may be able to achieve therapeutic efficacy with lower dosages
of each agent, thus reducing the potential for adverse side effects.
[0159] An antagonist of SYNT may be produced using methods which are generally known in
the art. In particular, purified SYNT may be used to produce antibodies or to screen
libraries of pharmaceutical agents to identify those which specifically bind SYNT.
Antibodies to SYNT may also be generated using methods that are well known in the
art. Such antibodies may include, but are not limited to, polyclonal, monoclonal,
chimeric, and single chain antibodies. Fab fragments, and fragments produced by a
Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation)
are generally preferred for therapeutic use.
[0160] For the production of antibodies, various hosts including goats, rabbits, rats, mice,
humans, and others may be immunized by injection with SYNT or with any fragment or
oligopeptide thereof which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response. Such adjuvants include,
but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin)
and
Corynebacterium parvum are especially preferable.
[0161] It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies
to SYNT have an amino acid sequence consisting of at least about 5 amino acids, and
generally will consist of at least about 10 amino acids. It is also preferable that
these oligopeptides, peptides, or fragments are identical to a portion of the amino
acid sequence of the natural protein. Short stretches of SYNT amino acids may be fused
with those of another protein, such as KLH, and antibodies to the chimeric molecule
may be produced.
[0162] Monoclonal antibodies to SYNT may be prepared using any technique which provides
for the production of antibody molecules by continuous cell lines in culture. These
include, but are not limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature
256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et
al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol.
Cell Biol. 62:109-120.)
[0163] In addition, techniques developed for the production of "chimeric antibodies," such
as the splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S.
et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.)
Alternatively, techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce SYNT-specific single chain
antibodies. Antibodies with related specificity, but of distinct idiotypic composition,
may be generated by chain shuffling from random combinatorial immunoglobulin libraries.
(See, e.g., Burton. D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
[0164] Antibodies may also be produced by inducing
in vivo production in the lymphocyte population or by screening immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in the literature. (See,
e.g., Orlandi. R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G.
et al. (1991) Nature 349:293-299.)
[0165] Antibody fragments which contain specific binding sites for SYNT may also be generated.
For example, such fragments include, but are not limited to, F(ab')
2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments
generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively,
Fab expression libraries may be constructed to allow rapid and easy identification
of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et
al. (1989) Science 246:1275-1281.)
[0166] Various immunoassays may be used for screening to identify antibodies having the
desired specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the measurement of
complex formation between SYNT and its specific antibody. A two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two non-interfering SYNT epitopes
is generally used, but a competitive binding assay may also be employed (Pound,
supra).
[0167] Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques
may be used to assess the affinity of antibodies for SYNT. Affinity is expressed as
an association constant, K
a, which is defined as the molar concentration of SYNT-antibody complex divided by
the molar concentrations of free antigen and free antibody under equilibrium conditions.
The K
a determined for a preparation of polyclonal antibodies, which are heterogeneous in
their affinities for multiple SYNT epitopes, represents the average affinity, or avidity,
of the antibodies for SYNT. The K
a, determined for a preparation of monoclonal antibodies, which are monospecific for
a particular SYNT epitope, represents a true measure of affinity. High-affinity antibody
preparations with K
a ranging from about 10
9 to 10
12 L/mole are preferred for use in immunoassays in which the SYNT-antibody complex must
withstand rigorous manipulations. Low-affinity antibody preparations with K
a, ranging from about 10
6 to 10
7 L/mole are preferred for use in immunopurification and similar procedures which ultimately
require dissociation of SYNT, preferably in active form, from the antibody (Catty,
D. (1988)
Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer ( 1991 )
A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
[0168] The titer and avidity of polyclonal antibody preparations may be further evaluated
to determine the quality and suitability of such preparations for certain downstream
applications. For example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed
in procedures requiring precipitation of SYNT-antibody complexes. Procedures for evaluating
antibody specificity, titer, and avidity, and guidelines for antibody quality and
usage in various applications, are generally available. (See, e.g., Catty,
supra, and Coligan et al.,
supra.)
[0169] In another embodiment of the invention, the polynucleotides encoding SYNT, or any
fragment or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene expression can be achieved by designing complementary sequences
or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding
or regulatory regions of the gene encoding SYNT. Such technology is well known in
the art, and antisense oligonucleotides or larger fragments can be designed from various
locations along the coding or control regions of sequences encoding SYNT. (See, e.g.,
Agrawal, S., ed. (1996)
Antisense Therapeutics, Humana Press Inc., Totawa NJ.)
[0170] In therapeutic use, any gene delivery system suitable for introduction of the antisense
sequences into appropriate target cells can be used. Antisense sequences can be delivered
intracellularly in the form of an expression plasmid which, upon transcription, produces
a sequence complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.. Slater. J.E. et al, (1998) J. Allergy Clin. Immunol, 102(3):469-475;
and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be
introduced intracellularly through the use of viral vectors, such as retrovirus and
adeno-associated virus vectors. (See, e.g.. Miller. A.D. (1990) Blood 76:271: Ausubel,
supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.) Other gene delivery
mechanisms include liposome-derived systems, artificial viral envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull. 51(1):217-225:
Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315: and Morris, M.C. et al.
(1997) Nucleic Acids Res. 25(14):2730-2736.)
[0171] In another embodiment of the invention, polynucleotides encoding SYNT may be used
for somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1
disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science
288:669-672), severe combined immunodeficiency syndrome associated with an inherited
adenosine deaminase (ADA) deficiency (Blaese, R.M. et al, (1995) Science 270:475-480;
Bordignon, C. et al, (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al.
(1993) Cell 75:207-216; Crystal. R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal,
R.G. et al, (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia,
and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R.G.
(1995) Science 270:404-410; Verma, I.M. and Somia, N. (1997) Nature 389:239-242)),
(ii) express a conditionally lethal gene product (e.g., in the case of cancers which
result from unregulated cell proliferation), or (iii) express a protein which affords
protection against intracellular parasites (e.g., against human retroviruses, such
as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla,
E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus
(HBV, HCV); fungal parasites, such as
Candida albicans and
Paracoccidioides brasiliensis; and protozoan parasites such as
Plasmodium falciparum and
Trypanosoma cruzi). In the case where a genetic deficiency in SYNT expression or regulation causes disease,
the expression of SYNT from an appropriate population of transduced cells may alleviate
the clinical manifestations caused by the genetic deficiency.
[0172] In a further embodiment of the invention, diseases or disorders caused by deficiencies
in SYNT are treated by constructing mammalian expression vectors encoding SYNT and
introducing these vectors by mechanical means into SYNT-deficient cells. Mechanical
transfer technologies for use with cells
in vivo or
ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold
particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993)
Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-5 10: Boulay, J-L. and
H. Récipon (1998) Curr. Opin. Biotechnol. 9:445-450).
[0173] Expression vectors that may be effective for the expression of SYNT include, but
are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). SYNT may be expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an
inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen. M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially
available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available
in the plasmids PVGRXR and FIND; Invitrogen); the FK506/rapamycin inducible promoter;
or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau,
supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene
encoding SYNT from a normal individual.
[0174] Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION
KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver
polynucleotides to target cells in culture and require minimal effort to optimize
experimental parameters. In the alternative, transformation is performed using the
calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or
by electroporation (Neumann, E. et al, (1982) EMBO J. 1:841-845), The introduction
of DNA to primary cells requires modification of these standardized mammalian transfection
protocols.
[0175] In another embodiment of the invention, diseases or disorders caused by genetic defects
with respect to SYNT expression are treated by constructing a retrovirus vector consisting
of (i) the polynucleotide encoding SYNT under the control of an independent promoter
or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging
signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus
cis-acting RNA sequences and coding sequences required for efficient vector propagation.
Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene)
and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
USA 92:6733-6737). incorporated by reference herein. The vector is propagated in an
appropriate vector producing cell line (VPCL) that expresses an envelope gene with
a tropism for receptors on the target cells or a promiscuous envelope protein such
as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987)
J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806;
Dull. T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol.
72:9873-9880). U.S. Patent Number 5.910,434 to Rigg ("Method for obtaining retrovirus
packaging cell lines producing high transducing efficiency retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated
by reference. Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4
+ T-cells), and the return of transduced cells to a patient are procedures well known
to persons skilled in the art of gene therapy and have been well documented (Ranga,
U. et al. (1997) J. Virol, 71:7020-7029; Bauer. G. et al, (1997) Blood 89:2259-2267;
Bonyhadi, M.L. (1997) J. Virol, 71:4707-4716: Ranga, U. et al. (1998) Proc. Natl.
Acad. Sci. USA 95:1201-1206; Su. L. (1997) Blood 89:2283-2290).
[0176] In the alternative, an adenovirus-based gene therapy delivery system is used to deliver
polynucleotides encoding SYNT to cells which have one or more genetic abnormalities
with respect to the expression of SYNT. The construction and packaging of adenovirus-based
vectors are well known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory
proteins into intact islets in the pancreas (Csete. M.E. et al. (1995) Transplantation
27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent Number
5.707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated
by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544; and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242,
both incorporated by reference herein.
[0177] In another alternative, a herpes-based, gene therapy delivery system is used to deliver
polynucleotides encoding SYNT to target cells which have one or more genetic abnormalities
with respect to the expression of SYNT. The use of herpes simplex virus (HSV)-based
vectors may be especially valuable for introducing SYNT to cells of the central nervous
system, for which HSV has a tropism. The construction and packaging of herpes-based
vectors are well known to those with ordinary skill in the art. A replication-competent
herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter
gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The
construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent
Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which
is hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant HSV d92 which consists of a genome containing at least one exogenous
gene to be transferred to a cell under the control of the appropriate promoter for
purposes including human gene therapy. Also taught by this patent are the construction
and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors,
see also Coins. W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev.
Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the growth
and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of ordinary skill in the art.
[0178] In another alternative, an alphavirus (positive, single-stranded RNA virus) vector
is used to deliver polynucleotides encoding SYNT to target cells. The biology of the
prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and
gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998)
Curr. Opin. Biotech. 9:464-469). During alphavirus RNA replication, a subgenomic RNA
is generated that normally encodes the viral capsid proteins. This subgenomic RNA
replicates to higher levels than the full-length genomic RNA, resulting in the overproduction
of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease
and polymerase). Similarly, inserting the coding sequence for SYNT into the alphavirus
genome in place of the capsid-coding region results in the production of a large number
of SYNT-coding RNAs and the synthesis of high levels of SYNT in vector transduced
cells. While alphavirus infection is typically associated with cell lysis within a
few days, the ability to establish a persistent infection in hamster normal kidney
cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication
of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga,
S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow
the introduction of SYNT into a variety of cell types. The specific transduction of
a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA transfections, and performing alphavirus infections, are well known to
those with ordinary skill in the art.
[0179] Oligonucleotides derived from the transcription initiation site, e.g., between about
positions - 10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology.
Triple helix pairing is useful because it causes inhibition of the ability of the
double helix to open sufficiently for the binding of polymerases, transcription factors,
or regulatory molecules. Recent therapeutic advances using triplex DNA have been described
in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr,
Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A complementary sequence or antisense
molecule may also be designed to block translation of mRNA by preventing the transcript
from binding to ribosomes.
[0180] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage
of RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically
and efficiently catalyze endonucleolytic cleavage of sequences encoding SYNT.
[0181] Specific ribozyme cleavage sites within any potential RNA target are initially identified
by scanning the target molecule for ribozyme cleavage sites, including the following
sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides, corresponding to the region of the target gene containing the
cleavage site, may be evaluated for secondary structural features which may render
the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated
by testing accessibility to hybridization with complementary oligonucleotides using
ribonuclease protection assays.
[0182] Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared
by any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated
by
in vitro and
in vivo transcription of DNA sequences encoding SYNT. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or
SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively
or inducibly, can be introduced into cell lines, cells, or tissues.
[0183] RNA molecules may be modified to increase intracellular stability and half-life.
Possible modifications include, but are not limited to, the addition of flanking sequences
at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl
rather than phosphodiesterase linkages within the backbone of the molecule. This concept
is inherent in the production of PNAs and can be extended in all of these molecules
by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine,
as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
[0184] An additional embodiment of the invention encompasses a method for screening for
a compound which is effective in altering expression of a polynucleotide encoding
SYNT. Compounds which may be effective in altering expression of a specific polynucleotide
may include, but are not limited to, oligonucleotides, antisense oligonucleotides,
triple helix-forming oligonucleotides, transcription factors and other polypeptide
transcriptional regulators, and non-macromolecular chemical entities which are capable
of interacting with specific polynucleotide sequences. Effective compounds may alter
polynucleotide expression by acting as either inhibitors or promoters of polynucleotide
expression. Thus, in the treatment of disorders associated with increased SYNT expression
or activity, a compound which specifically inhibits expression of the polynucleotide
encoding SYNT may be therapeutically useful, and in the treament of disorders associated
with decreased SYNT expression or activity, a compound which specifically promotes
expression of the polynucleotide encoding SYNT may be therapeutically useful.
[0185] At least one, and up to a plurality, of test compounds may be screened for effectiveness
in altering expression of a specific polynucleotide. A test compound may be obtained
by any method commonly known in the art, including chemical modification of a compound
known to be effective in altering polynucleotide expression: selection from an existing,
commercially-available or proprietary library of naturally-occurring or non-natural
chemical compounds; rational design of a compound based on chemical and/or structural
properties of the target polynucleotide; and selection from a library of chemical
compounds created combinatorially or randomly. A sample comprising a polynucleotide
encoding SYNT is exposed to at least one test compound thus obtained. The sample may
comprise, for example, an intact or permeabilized cell, or an
in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a
polynucleotide encoding SYNT are assayed by any method commonly known in the art.
Typically, the expression of a specific nucleotide is detected by hybridization with
a probe having a nucleotide sequence complementary to the sequence of the polynucleotide
encoding SYNT. The amount of hybridization may be quantified, thus forming the basis
for a comparison of the expression of the polynucleotide both with and without exposure
to one or more test compounds. Detection of a change in the expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in altering
the expression of the polynucleotide. A screen for a compound effective in altering
expression of a specific polynucleotide can be carried out, for example, using a
Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt,
G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell
(Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular
embodiment of the present invention involves screening a combinatorial library of
oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids,
and modified oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5.686.242; Bruice, T.W. et al.
(2000) U.S. Patent No. 6.022,691).
[0186] Many methods for introducing vectors into cells or tissues are available and equally
suitable for use
in vivo, in vitro, and
ex vivo. For
ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally
propagated for autologous transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be achieved using methods
which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol.
15:462-466.)
[0187] Any of the therapeutic methods described above may be applied to any subject in need
of such therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and monkeys.
[0188] An additional embodiment of the invention relates to the administration of a pharmaceutical
composition which generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins. Various formulations are commonly known and are thoroughly discussed
in the latest edition of Remington's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such pharmaceutical compositions may consist of SYNT,
antibodies to SYNT, and mimetics, agonists, antagonists, or inhibitors of SYNT.
[0189] The pharmaceutical compositions utilized in this invention may be administered by
any number of routes including, but not limited to, oral, intravenous, intramuscular,
intra-arterial. intramedullary, intrathecal, intraventricular, pulmonary, transdermal,
subcutaneous, intraperitoneal. intranasal, enteral, topical, sublingual, or rectal
means.
[0190] Pharmaceutical compositions for pulmonary administration may be prepared in liquid
or dry powder form. These compositions are generally aerosolized immediately prior
to inhalation by the patient. In the case of small molecules (e.g. traditional low
molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known
in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent
developments in the field of pulmonary delivery via the alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood circulation
(see. e.g., Patton, J.S. et al., U.S. Patent No. 5.997,848). Pulmonary delivery has
the advantage of administration without needle injection, and obviates the need for
potentially toxic penetration enhancers.
[0191] Pharmaceutical compositions suitable for use in the invention include compositions
wherein the active ingredients are contained in an effective amount to achieve the
intended purpose. The determination of an effective dose is well within the capability
of those skilled in the art.
[0192] Specialized forms of pharmaceutical compositions may be prepared for direct intracellular
delivery of macromolecules comprising SYNT or fragments thereof. For example, liposome
preparations containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the macromolecule. Alternatively, SYNT or a fragment thereof
may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion
proteins thus generated have been found to transduce into the cells of all tissues,
including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science
285:1569-1572).
[0193] For any compound, the therapeutically effective dose can be estimated initially either
in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine
the appropriate concentration range and route of administration. Such information
can then be used to determine useful doses and routes for administration in humans.
[0194] A therapeutically effective dose refers to that amount of active ingredient, for
example SYNT or fragments thereof, antibodies of SYNT, and agonists, antagonists or
inhibitors of SYNT, which ameliorates the symptoms or condition. Therapeutic efficacy
and toxicity may be determined by standard pharmaceutical procedures in cell cultures
or with experimental animals, such as by calculating the ED
50 (the dose therapeutically effective in 50% of the population) or LD
50 (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the therapeutic index, which can be expressed as the LD
50/ED
50 ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies are used to formulate
a range of dosage for human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that includes the ED
50 with little or no toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route of administration.
[0195] The exact dosage will be determined by the practitioner, in light of factors related
to the subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active moiety or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease state, the general
health of the subject, the age, weight, and gender of the subject, time and frequency
of administration, drug combinations), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every
week, or biweekly depending on the half-life and clearance rate of the particular
formulation.
[0196] Normal dosage amounts may vary from about 0.1
µg to 100,000
µg, up to a total dose of about 1 gram, depending upon the route of administration.
Guidance as to particular dosages and methods of delivery is provided in the literature
and generally available to practitioners in the art. Those skilled in the art will
employ different formulations for nucleotides than for proteins or their inhibitors.
Similarly, delivery of polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
[0197] In another embodiment, antibodies which specifically bind SYNT may be used for the
diagnosis of disorders characterized by expression of SYNT, or in assays to monitor
patients being treated with SYNT or agonists, antagonists, or inhibitors of SYNT.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described
above for therapeutics. Diagnostic assays for SYNT include methods which utilize the
antibody and a label to detect SYNT in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification, and may be labeled
by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter
molecules, several of which are described above, are known in the art and may be used.
[0198] A variety of protocols for measuring SYNT, including ELISAs, RIAs, and FACS, are
known in the art and provide a basis for diagnosing altered or abnormal levels of
SYNT expression. Normal or standard values for SYNT expression are established by
combining body fluids or cell extracts taken from normal mammalian subjects, for example,
human subjects, with antibody to SYNT under conditions suitable for complex formation.
The amount of standard complex formation may be quantitated by various methods, such
as photometric means. Quantities of SYNT expressed in subject, control, and disease
samples from biopsied tissues are compared with the standard values. Deviation between
standard and subject values establishes the parameters for diagnosing disease.
[0199] In another embodiment of the invention, the polynucleotides encoding SYNT may be
used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules. and PNAs. The polynucleotides may
be used to detect and quantify gene expression in biopsied tissues in which expression
of SYNT may be correlated with disease. The diagnostic assay may be used to determine
absence, presence, and excess expression of SYNT, and to monitor regulation of SYNT
levels during therapeutic intervention.
[0200] In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide
sequences, including genomic sequences, encoding SYNT or closely related molecules
may be used to identify nucleic acid sequences which encode SYNT. The specificity
of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory
region, or from a less specific region, e.g., a conserved motif, and the stringency
of the hybridization or amplification will determine whether the probe identifies
only naturally occurring sequences encoding SYNT, allelic variants, or related sequences.
[0201] Probes may also be used for the detection of related sequences, and may have at least
50% sequence identity to any of the SYNT encoding sequences. The hybridization probes
of the subject invention may be DNA or RNA and may be derived from the sequence of
SEQ ID NO: 16-30 or from genomic sequences including promoters, enhancers, and introns
of the SYNT gene.
[0202] Means for producing specific hybridization probes for DNAs encoding SYNT include
the cloning of polynucleotide sequences encoding SYNT or SYNT derivatives into vectors
for the production of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes
in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled
nucleotides. Hybridization probes may be labeled by a variety of reporter groups,
for example, by radionuclides such as
32P or
35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0203] Polynucleotide sequences encoding SYNT may be used for the diagnosis of disorders
associated with expression of SYNT. Examples of such disorders include, but are not
limited to, an immune disorder such as inflammation, actinic keratosis, acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis. Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis, glomerulonephritis. Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable
bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue
disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis,
psoriasis. Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications
of cancer, hemodialysis, and extracorporeal circulation, trauma, viral. bacterial,
fungal, parasitic, protozoal, and helminthic infections, and hematopoietic cancer
including lymphoma, leukemia, and myeloma; a neuronal disorder, such as akathesia,
Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia,
cerebral neoplasms, dementia, depression, diabetic neuropathy, Down's syndrome, tardive
dyskinesia, dystonias, epilepsy, Huntington's disease, peripheral neuropathy, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpetic
neuralgia, schizophrenia, and Tourette's disorder; a reproductive disorder, such as
a disorder of prolactin production, infertility, including tuba) disease, ovulatory
defects, and endometriosis, a disruption of the estrous cycle, a disruption of the
menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an
endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic
pregnancy, and teratogenesis; cancer of the breast, fibrocystic breast disease, and
galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of
the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and gynecomastia; and a cell proliferative
disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. The polynucleotide
sequences encoding SYNT may be used in Southern or northern analysis, dot blot, or
other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat
ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to
detect altered SYNT expression. Such qualitative or quantitative methods are well
known in the art.
[0204] In a particular aspect, the nucleotide sequences encoding SYNT may be useful in assays
that detect the presence of associated disorders, particularly those mentioned above.
The nucleotide sequences encoding SYNT may be labeled by standard methods and added
to a fluid or tissue sample from a patient under conditions suitable for the formation
of hybridization complexes. After a suitable incubation period, the sample is washed
and the signal is quantified and compared with a standard value. If the amount of
signal in the patient sample is significantly altered in comparison to a control sample
then the presence of altered levels of nucleotide sequences encoding SYNT in the sample
indicates the presence of the associated disorder. Such assays may also be used to
evaluate the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to monitor the treatment of an individual patient.
[0205] In order to provide a basis for the diagnosis of a disorder associated with expression
of SYNT, a normal or standard profile for expression is established. This may be accomplished
by combining body fluids or cell extracts taken from normal subjects, either animal
or human, with a sequence, or a fragment thereof, encoding SYNT, under conditions
suitable for hybridization or amplification. Standard hybridization may be quantified
by comparing the values obtained from normal subjects with values from an experiment
in which a known amount of a substantially purified polynucleotide is used. Standard
values obtained in this manner may be compared with values obtained from samples from
patients who are symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0206] Once the presence of a disorder is established and a treatment protocol is initiated,
hybridization assays may be repeated on a regular basis to determine if the level
of expression in the patient begins to approximate that which is observed in the normal
subject. The results obtained from successive assays may be used to show the efficacy
of treatment over a period ranging from several days to months.
[0207] With respect to cancer, the presence of an abnormal amount of transcript (either
under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition
for the development of the disease, or may provide a means for detecting the disease
prior to the appearance of actual clinical symptoms. A more definitive diagnosis of
this type may allow health professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further progression of the
cancer.
[0208] Additional diagnostic uses for oligonucleotides designed from the sequences encoding
SYNT may involve the use of PCR. These oligomers may be chemically synthesized, generated
enzymatically, or produced
in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding SYNT, or
a fragment of a polynucleotide complementary to the polynucleotide encoding SYNT,
and will be employed under optimized conditions for identification of a specific gene
or condition. Oligomers may also be employed under less stringent conditions for detection
or quantification of closely related DNA or RNA sequences.
[0209] In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences
encoding SYNT may be used to detect single nucleotide polymorphisms (SNPs). SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited or
acquired genetic disease in humans. Methods of SNP detection include, but are not
limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP
(fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide
sequences encoding SYNT are used to amplify DNA using-the polymerase chain reaction
(PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy
samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary
and tertiary structures of PCR products in single-stranded form, and these differences
are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide
primers are fluorescently labeled, which allows detection of the amplimers in high-throughput
equipment such as DNA sequencing machines. Additionally, sequence database analysis
methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by
comparing the sequence of individual overlapping DNA fragments which assemble into
a common consensus sequence. These computer-based methods filter out sequence variations
due to laboratory preparation of DNA and sequencing errors using statistical models
and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may
be detected and characterized by mass spectrometry using, for example, the high throughput
MASSARRAY system (Sequenom, Inc., San Diego CA).
[0210] Methods which may also be used to quantify the expression of SYNT include radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244: Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation
of multiple samples may be accelerated by running the assay in a high-throughput format
where the oligomer or polynucleotide of interest is presented in various dilutions
and a spectrophotometric or colorimetric response gives rapid quantitation.
[0211] In further embodiments, oligonucleotides or longer fragments derived from any of
the polynucleotide sequences described herein may be used as elements on a microarray.
The microarray can be used in transcript imaging techniques which monitor the relative
expression levels of large numbers of genes simultaneously as described in Seilhamer,
J.J. et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, incorporated
herein by reference. The microarray may also be used to identify genetic variants,
mutations, and polymorphisms. This information may be used to determine gene function,
to understand the genetic basis of a disorder, to diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression, and to develop
and monitor the activities of therapeutic agents in the treatment of disease. In particular,
this information may be used to develop a pharmacogenomic profile of a patient in
order to select the most appropriate and effective treatment regimen for that patient.
For example, therapeutic agents which are highly effective and display the fewest
side effects may be selected for a patient based on his/her pharmacogenomic profile.
[0212] In another embodiment, antibodies specific for SYNT, or SYNT or fragments thereof
may be used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein interactions, drug-target interactions, and gene expression
profiles, as described above.
[0213] A particular embodiment relates to the use of the polynucleotides of the present
invention to generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of gene expression by a particular tissue or cell type.
Global gene expression patterns are analyzed by quantifying the number of expressed
genes and their relative abundance under given conditions and at a given time. (See
Seilhamer et al., "Comparative Gene Transcript Analysis." U.S. Patent Number 5,840,484,
expressly incorporated by reference herein.) Thus a transcript image may be generated
by hybridizing the polynucleotides of the present invention or their complements to
the totality of transcripts or reverse transcripts of a particular tissue or cell
type. In one embodiment, the hybridization takes place in high-throughput format,
wherein the polynucleotides of the present invention or their complements comprise
a subset of a plurality of elements on a microarray. The resultant transcript image
would provide a profile of gene activity.
[0214] Transcript images may be generated using transcripts isolated from tissues, cell
lines, biopsies, or other biological samples. The transcript image may thus reflect
gene expression
in vivo, as in the case of a tissue or biopsy sample, or
in vitro, as in the case of a cell line.
[0215] Transcript images which profile the expression of the polynucleotides of the present
invention may also be used in conjunction with
in vitro model systems and preclinical evaluation of pharmaceuticals. as well as toxicological
testing of industrial and naturally-occurring environmental compounds. All compounds
induce characteristic gene expression patterns, frequently termed molecular fingerprints
or toxicant signatures, which are indicative of mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a signature similar to that of a compound with known toxicity,
it is likely to share those toxic properties. These fingerprints or signatures are
most useful and refined when they contain expression information from a large number
of genes and gene families. Ideally, a genome-wide measurement of expression provides
the highest quality signature. Even genes whose expression is not altered by any tested
compounds are important as well, as the levels of expression of these genes are used
to normalize the rest of the expression data. The normalization procedure is useful
for comparison of expression data after treatment with different compounds. While
the assignment of gene function to elements of a toxicant signature aids in interpretation
of toxicity mechanisms, knowledge of gene function is not necessary for the statistical
matching of signatures which leads to prediction of toxicity. (See, for example, Press
Release 00-02 from the National Institute of Environmental Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore,
it is important and desirable in toxicological screening using toxicant signatures
to include all expressed gene sequences.
[0216] In one embodiment, the toxicity of a test compound is assessed by treating a biological
sample containing nucleic acids with the test compound. Nucleic acids that are expressed
in the treated biological sample are hybridized with one or more probes specific to
the polynucleotides of the present invention, so that transcript levels corresponding
to the polynucleotides of the present invention may be quantified. The transcript
levels in the treated biological sample are compared with levels in an untreated biological
sample. Differences in the transcript levels between the two samples are indicative
of a toxic response caused by the test compound in the treated sample.
[0217] Another particular embodiment relates to the use of the polypeptide sequences of
the present invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global pattern of protein expression in a particular tissue or cell
type. Each protein component of a proteome can be subjected individually to further
analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the
number of expressed proteins and their relative abundance under given conditions and
at a given time. A profile of a cell's proteome may thus be generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one embodiment,
the separation is achieved using two-dimensional gel electrophoresis, in which proteins
from a sample are separated by isoelectric focusing in the first dimension, and then
according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in
the second dimension (Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely positioned spots,
typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical density of each protein spot is generally proportional to the
level of the protein in the sample. The optical densities of equivalently positioned
protein spots from different samples, for example, from biological samples either
treated or untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein spot density related to the treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing chemical
or enzymatic cleavage followed by mass spectrometry. The identity of the protein in
a spot may be determined by comparing its partial sequence, preferably of at least
5 contiguous amino acid residues, to the polypeptide sequences of the present invention.
In some cases, further sequence data may be obtained for definitive protein identification.
[0218] A proteomic profile may also be generated using antibodies specific for SYNT to quantify
the levels of SYNT expression. In one embodiment, the antibodies are used as elements
on a microarray, and protein expression levels are quantified by exposing the microarray
to the sample and detecting the levels of protein bound to each array element (Lueking,
A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et at. (1999) Biotechniques
27:778-788). Detection may be performed by a variety of methods known in the art,
for example, by reacting the proteins in the sample with a thiol- or amino-reactive
fluorescent compound and detecting the amount of fluorescence bound at each array
element.
[0219] Toxicant signatures at the proteome level are also useful for toxicological screening,
and should be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation between transcript and protein abundances for some proteins
in some tissues (Anderson. N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537),
so proteome toxicant signatures may be useful in the analysis of compounds which do
not significantly affect the transcript image, but which alter the proteomic profile.
In addition, the analysis of transcripts in body fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and informative in
such cases.
[0220] In another embodiment, the toxicity of a test compound is assessed by treating a
biological sample containing proteins with the test compound. Proteins that are expressed
in the treated biological sample are separated so that the amount of each protein
can be quantified. The amount of each protein is compared to the amount of the corresponding
protein in an untreated biological sample. A difference in the amount of protein between
the two samples is indicative of a toxic response to the test compound in the treated
sample. Individual proteins are identified by sequencing the amino acid residues of
the individual proteins and comparing these partial sequences to the polypeptides
of the present invention.
[0221] In another embodiment, the toxicity of a test compound is assessed by treating a
biological sample containing proteins with the test compound. Proteins from the biological
sample are incubated with antibodies specific to the polypeptides of the present invention.
The amount of protein recognized by the antibodies is quantified. The amount of protein
in the treated biological sample is compared with the amount in an untreated biological
sample. A difference in the amount of protein between the two samples is indicative
of a toxic response to the test compound in the treated sample.
[0222] Microarrays may be prepared, used, and analyzed using methods known in the art. (See,
e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996)
Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon. D. et al. (1995) PCT application WO95/35505; Heller, R.A. et
al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997)
U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly
described in
DNA Microarrays: A Practical Approach, M. Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated
by reference.
[0223] In another embodiment of the invention, nucleic acid sequences encoding SYNT may
be used to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may be preferable over coding sequences. For example, conservation
of a coding sequence among members of a multi-gene family may potentially cause undesired
cross hybridization during chromosomal mapping. The sequences may be mapped to a particular
chromosome, to a specific region of a chromosome, or to artificial chromosome constructions,
e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial
artificial chromosomes (BACs), bacterial P1 constructions. or single chromosome cDNA
libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355: Price.
C.M. (1993) Blood Rev. 7:127-134: and Trask. B.J. (1991) Trends Genet. 7:149-154.)
Once mapped, the nucleic acid sequences of the invention may be used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state with
the inheritance of a particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, e.g., Lander, E.S. and D. Botstein ( 1986) Proc. Natl, Acad. Sci. USA
83:7353-7357.)
[0224] Fluorescent
in situ hybridization (FISH) may be correlated with other physical and genetic map data.
(See, e.g., Heinz-Ulrich, et al, (1995) in Meyers,
supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals
or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation
between the location of the gene encoding SYNT on a physical map and a specific disorder,
or a predisposition to a specific disorder, may help define the region of DNA associated
with that disorder and thus may further positional cloning efforts.
[0225] In situ hybridization of chromosomal preparations and physical mapping techniques, such as
linkage analysis using established chromosomal markers, may be used for extending
genetic maps. Often the placement of a gene on the chromosome of another mammalian
species, such as mouse, may reveal associated markers even if the exact chromosomal
locus is not known. This information is valuable to investigators searching for disease
genes using positional cloning or other gene discovery techniques. Once the gene or
genes responsible for a disease or syndrome have been crudely localized by genetic
linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23. any
sequences mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant invention may also be used to detect differences in the chromosomal
location due to translocation, inversion, etc., among normal, carrier, or affected
individuals.
[0226] In another embodiment of the invention, SYNT, its catalytic or immunogenic fragments,
or oligopeptides thereof can be used for screening libraries of compounds in any of
a variety of drug screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell surface, or located
intracellularly. The formation of binding complexes between SYNT and the agent being
tested may be measured.
[0227] Another technique for drug screening provides for high throughput screening of compounds
having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et
al. (1984) PCT application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate. The test compounds are
reacted with SYNT, or fragments thereof. and washed. Bound SYNT is then detected by
methods well known in the art. Purified SYNT can also be coated directly onto plates
for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing
antibodies can be used to capture the peptide and immobilize it on a solid support.
[0228] In another embodiment, one may use competitive drug screening assays in which neutralizing
antibodies capable-of binding SYNT specifically compete with a test compound for binding
SYNT. In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more antigenic determinants with SYNT.
[0229] In additional embodiments. the nucleotide sequences which encode SYNT may be used
in any molecular biology techniques that have yet to be developed, provided the new
techniques rely on properties of nucleotide sequences that are currently known, including,
but not limited to, such properties as the triplet genetic code and specific base
pair interactions.
[0230] Without further elaboration, it is believed that one skilled in the art can, using
the preceding description, utilize the present invention to its fullest extent. The
following preferred specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
[0231] The disclosures of all patents, applications, and publications mentioned above and
below, in particular U.S. Ser. No. 60/144,992 and U.S. Ser. No. 60/168,858 are hereby
expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
[0232] RNA was purchased from Clontech or isolated from tissues described in Table 4. Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate.
The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform.
RNA was precipitated from the lysates with either isopropanol or sodium acetate and
ethanol, or by other routine methods.
[0233] Phenol extraction and precipitation of RNA were repeated as necessary to increase
RNA purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+)
RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation
kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
[0234] In some cases, Stratagene was provided with RNA and constructed the corresponding
cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997,
supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers.
Synthetic oligonucleotide adapters were ligated to double stranded cDNA. and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries,
the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or
SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose
gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of
the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1
plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY
plasmid (Incyte Genomics, Palo Alto CA). Recombinant plasmids were transformed into
competent
E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α. DH10B, or
ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
[0235] Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were
purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega): an AGTC Miniprep purification kit (Edge Biosystems. Gaithersburg
MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification
systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation,
plasmids were resuspended in 0.1 ml of distilled water and stored, with or without
lyophilization, at 4°C.
[0236] Alternatively, plasmid DNA was amplified from host cell lysates using direct link
PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal cycling steps were carried out in a single reaction mixture. Samples
were processed and stored in 384-well plates, and the concentration of amplified plasmid
DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene
OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
[0237] Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific)
or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in
ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready
reaction kit (PE Biosystems). Electrophoretic separation of cDNA sequencing reactions
and detection of labeled polynucleotides were carried out using the MEGABACE 1000
DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system
(PE Biosystems) in conjunction with standard ABI protocols and base calling software;
or other sequence analysis systems known in the art. Reading frames within the cDNA
sequences were identified using standard methods (reviewed in Ausubel. 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques
disclosed in Example VI.
[0238] The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed
using a combination of software programs which utilize algorithms well known to those
skilled in the art. Table 5 summarizes the tools, programs, and algorithms used and
provides applicable descriptions, references, and threshold parameters. The first
column of Table 5 shows the tools. programs, and algorithms used, the second column
provides brief descriptions thereof, the third column presents appropriate references,
all of which are incorporated by reference herein in their entirety, and the fourth
column presents, where applicable, the scores, probability values. and other parameters
used to evaluate the strength of a match between two sequences (the higher the score,
the greater the homology between two sequences). Sequences were analyzed using MACDNASIS
PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default parameters specified by the clustal algorithm as incorporated into
the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the
percent identity between aligned sequences.
[0239] The polynucleotide sequences were validated by removing vector, linker, and polyA
sequences and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a selection of public databases such as the GenBank primate,
rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO,
PRODOM, and PFAM to acquire annotation using programs based on BLAST, PASTA, and BLIMPS.
The sequences were assembled into full length polynucleotide sequences using programs
based on Phred, Phrap, and Consed, and were screened for open reading frames using
programs based on GeneMark, BLAST, and PASTA. The full length polynucleotide sequences
were translated to derive the corresponding full length amino acid sequences, and
these full length sequences were subsequently analyzed by querying against databases
such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO,
PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary structures
of gene families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
[0240] The programs described above for the assembly and analysis of full length polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence fragments
from SEQ ID NO: 16-30. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization and amplification technologies were described in The Invention
section above.
IV. Analysis of Polynucleotide Expression
[0241] Northern analysis is a laboratory technique used to detect the presence of a transcript
of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a particular cell type or tissue have been bound. (See. e.g., Sambrook,
supra, ch. 7: Ausubel. 1995,
supra, ch. 4 and 16.)
[0242] Analogous computer techniques applying BLAST were used to search for identical or
related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics).
This analysis is much faster than multiple membrane-based hybridizations. In addition,
the sensitivity of the computer search can be modified to determine whether any particular
match is categorized as exact or similar. The basis of the search is the product score,
which is defined as:

The product score takes into account both the degree of similarity between two sequences
and the length of the sequence match. The product score is a normalized value between
0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent
nucleotide identity and the product is divided by (5 times the length of the shorter
of the two sequences). The BLAST score is calculated by assigning a score of +5 for
every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch.
Two sequences may share more than one HSP (separated by gaps). If there is more than
one HSP, then the pair with the highest BLAST score is used to calculate the product
score. The product score represents a balance between fractional overlap and quality
in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared.
A product score of 70 is produced either by 100% identity and 70% overlap at one end,
or by 88% identity and 100% overlap at the other. A product score of 50 is produced
either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
[0243] The results of northern analyses are reported as a percentage distribution of libraries
in which the transcript encoding SYNT occurred. Analysis involved the categorization
of cDNA libraries by organ/tissue and disease. The organ/tissue categories included
cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories
included cancer. inflammation, trauma, cell proliferation, neurological, and pooled.
For each category, the number of libraries expressing the sequence of interest was
counted and divided by the total number of libraries across all categories. Percentage
values of tissue-specific and disease- or condition-specific expression are reported
in Table 3.
V. Extension of SYNT Encoding Polynucleotides
[0244] The full length nucleic acid sequences of SEQ ID NO:16-30 were produced by extension
of an appropriate fragment of the full length molecule using oligonucleotide primers
designed from this fragment. One primer was synthesized to initiate 5' extension of
the known fragment, and the other primer, to initiate 3'extension of the known fragment.
The initial primers were designed using OLIGO 4.06 software (National Biosciences),
or another appropriate program, to be about 22 to 30 nucleotides in length, to have
a GC content of about 50% or more, and to anneal to the target sequence at temperatures
of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
[0245] Selected human cDNA libraries were used to extend the sequence. If more than one
extension was necessary or desired, additional or nested sets of primers were designed.
[0246] High fidelity amplification was obtained by PCR using methods well known in the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction
buffer containing Mg
2+, (NH
4)
2SO
4, and β-mercaptoethanol. Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer pair PCI A and PCI B: Step 1: 94°C. 3 min; Step 2: 94°C, 15
sec: Step 3: 60°C, 1 min: Step 4: 68°C, 2 min: Step 5: Steps 2, 3, and 4 repeated
20 times; Step 6: 68°C. 5 min: Step 7: storage at 4°C. In the 5 alternative, the parameters
for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min: Step 2: 94°C. 15
sec; Step 3: 57°C, 1 min: Step 4: 68°C. 2 min; Step 5: Steps 2, 3, and 4 repeated
20 times; Step 6: 68°C. 5 min; Step 7: storage at 4°C.
[0247] The concentration of DNA in each well was determined by dispensing 100 µl PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved
in 1X TE and 0.5 µl of undiluted PCR product into each well of an opaque fluorimeter
plate (Coming Costar, Acton MA), allowing the DNA to bind to the reagent. The plate
was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence
of the sample and to quantify the concentration of DNA. A5 µl to 10 µl aliquot of
the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to
determine which reactions were successful in extending the sequence.
[0248] The extended nucleotides were desalted and concentrated, transferred to 384-well
plates. digested with CviJI cholera virus endonuclease (Molecular Biology Research.
Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated
on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar
digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs. Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated
with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected
into competent
E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual
colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb
liquid media.
[0249] The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham
Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters:
Step 1: 94°C. 3 min: Step 2: 94°C. 15 sec: Step 3: 60°C, 1 min: Step 4: 72°C, 2 min:
Step 5: steps 2. 3, and 4 repeated 29 times: Step 6: 73°C, 5 min: Step 7: storage
at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above.
Samples with low DNA recoveries were reamplified using the same conditions as described
above. Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using
DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham
Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction
kit (PE Biosystems).
[0250] In like manner, the polynucleotide sequences of SEQ ID NO: 16-30 are used to obtain
5' regulatory sequences using the procedure above, along with oligonucleotides designed
for such extension, and an appropriate genomic library.
V. Chromosomal Mapping of SNYT Encoding Polynucleotides
[0251] The cDNA sequences which were used to assemble SEQ ID NO:16-30 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using BLAST
and other implementations of the Smith-Waterman algorithm. Sequences from these databases
that matched SEQ ID NO:16-30 were assembled into clusters of contiguous and overlapping
sequences using assembly algorithms such as Phrap (Table 5). Radiation hybrid and
genetic mapping data available from public resources such as the Stanford Human Genome
Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Généthon were used
to determine if any of the clustered sequences had been previously mapped. Inclusion
of a mapped sequence in a cluster resulted in the assignment of all sequences of that
cluster, including its particular SEQ ID NO:, to that map location.
[0252] The genetic map locations of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18. SEQ ID NO:21,
SEQ ID NO:24. SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28. SEQ ID NO:29, and SEQ ID NO:30
are described in The Invention as ranges, or intervals, of human chromosomes. More
than one map location is reported for SEQ ID NO:28 and SEQ ID NO:29, indicating that
previously mapped sequences having similarity, but not complete identity, to SEQ ID
NO:28 and SEQ ID NO:29 were assembled into their respective clusters. The map position
of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's
p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies
between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase
(Mb) of DNA in humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances are based on genetic markers mapped by Généthon which
provide boundaries for radiation hybrid markers whose sequences were included in each
of the clusters. Diseases associated with the public and Incyte sequences located
within the indicated intervals are also reported in the Invention where applicable.
Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99"
World Wide Web site which can be accessed at http://www.ncbi.nlm.nih.gov/genemap,
can be employed to determine if previously identified disease genes map within or
in proximity to the intervals indicated above.
VI. Labeling and Use of Individual Hybridization Probes
[0253] Hybridization probes derived from SEQ ID NO:16-30 are employed to screen cDNAs, genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20
base pairs, is specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art
software such as OLIGO 4.06 software (National Biosciences) and labeled by combining
50 pmol of each oligomer. 250 µCi of [γ-
32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using
a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10
7 counts per minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human genomic DNA digested with one of the following endonucleases: Ase
I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
[0254] The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to
nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried
out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed
at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate
and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography
or an alternative imaging means and compared.
VII. Microarrays
[0255] The linkage or synthesis of array elements upon a microarray can be achieved utilizing
photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler,
supra), mechanical microspotting technologies, and derivatives thereof. The substrate in
each of the aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used
to arrange and link elements to the surface of a substrate using thermal, UV, chemical,
or mechanical bonding procedures. A typical array may be produced using available
methods and machines well known to those of ordinary skill in the art and may contain
any appropriate number of elements. (See, e.g., Schena, M. et al.(1995) Science 270:467-470;
Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall. A. and J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
[0256] Full length cDNAs. Expressed Sequence Tags (ESTs), or fragments or oligomers thereof
may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization
can be selected using software well known in the art such as LASERGENE software (DNASTAR).
The array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the biological sample are conjugated to a fluorescent label or
other molecular tag for ease of detection. After hybridization, nonhybridized nucleotides
from the biological sample are removed. and a fluorescence scanner is used to detect
hybridization at each array element. Alternatively, laser desorbtion and mass spectrometry
may be used for detection of hybridization. The degree of complementarity and the
relative abundance of each polynucleotide which hybridizes to an element on the microarray
may be assessed. In one embodiment, microarray preparation and usage is described
in detail below.
Tissue or Cell Sample Preparation
[0257] Total RNA is isolated from tissue samples using the guanidinium thiocyanate method
and poly(A)
+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)
+ RNA sample is reverse transcribed using MMLV reverse-transcriptase. 0.05 pg/µl oligo-(dT)
primer (21mer), 1X first strand buffer, 0.03 units/µl RNase inhibitor, 500µM dATP,
500 µM dGTP, 500 µM dTTP, 40 µM dCTP, 40 µM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia
Biotech). The reverse transcription reaction is performed in a 25 ml volume containing
200 ng poly(A)
+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)
+ RNAs are synthesized by
in vitro transcription from non-coding yeast genomic DNA. After incubation at 37°C for 2 hr,
each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with
2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85°C to the stop the
reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN
30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto
CA) and after combining, both reaction samples are ethanol precipitated using 1ml
of glycogen (1mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample
is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY)
and resuspended in 14 µl 5X SSC/0.2% SDS.
Microarray Preparation
[0258] Sequences of the present invention are used to generate array elements. Each array
element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the
cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial
quantity of 1-2 ng to a final quantity greater than 5 µg. Amplified array elements
are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
[0259] Purified array elements are immobilized on polymer-coated glass slides. Glass microscope
slides (Coming) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive
distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed
extensively in distilled water. and coated with 0.05% aminopropyl silane (Sigma) in
95% ethanol. Coated slides are cured in a 110°C oven.
[0260] Array elements are applied to the coated glass substrate using a procedure described
in US Patent No. 5.807.522. incorporated herein by reference. 1µl of the array element
DNA, at an average concentration of 100 ng/µl, is loaded into the open capillary printing
element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl
of array element sample per slide.
[0261] Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled
water. Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes
at 60°C followed by washes in 0.2% SDS and distilled water as before.
Hybridization
[0262] Hybridization reactions contain 9 µl of sample mixture consisting of 0.2 µg each
of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65°C for 5 minutes and is aliquoted onto the microarray
surface and covered with an 1.8 cm
2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity internally
by the addition of 140 µl of 5X SSC in a comer of the chamber. The chamber containing
the arrays is incubated for about 6.5 hours at 60°C. The arrays are washed for 10
min at 45°C in a first wash buffer (1 X SSC. 0.1% SDS), three times for 10 minutes
each at 45°C in a second wash buffer (0.1X SSC), and dried.
Detection
[0263] Reporter-labeled hybridization complexes are detected with a microscope equipped
with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation
of Cy5. The excitation laser light is focused on the array using a 20X microscope
objective (Nikon, Inc., Melville NY). The slide containing the array is placed on
a computer-controlled X-Y stage on the microscope and raster-scanned past the objective.
The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution
of 20 micrometers.
[0264] In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors
(PMT R1477, Hamamatsu Photonics Systems. Bridgewater NJ) corresponding to the two
fluorophores. Appropriate filters positioned between the array and the photomultiplier
tubes are used to filter the signals. The emission maxima of the fluorophores used
are 565 nm for Cy3 and 650 nm for Cy5. Each array is typically scanned twice, one
scan per fluorophore using the appropriate filters at the laser source, although the
apparatus is capable of recording the spectra from both fluorophores simultaneously.
[0265] The sensitivity of the scans is typically calibrated using the signal intensity generated
by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the
intensity of the signal at that location to be correlated with a weight ratio of hybridizing
species of 1:100,000. When two samples from different sources (e.g., representing
test and control cells), each labeled with a different fluorophore, are hybridized
to a single array for the purpose of identifying genes that are differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores
and adding identical amounts of each to the hybridization mixture.
[0266] The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible
PC computer. The digitized data are displayed as an image where the signal intensity
is mapped using a linear 20-color transformation to a pseudocolor scale ranging from
blue (low signal) to red (high signal). The data is also analyzed quantitatively.
Where two different fluorophores are excited and measured simultaneously, the data
are first corrected for optical crosstalk (due to overlapping emission spectra) between
the fluorophores using each fluorophore' emission spectrum.
[0267] A grid is superimposed over the fluorescence signal image such that the signal from
each spot is centered in each element of the grid. The fluorescence signal within
each element is then integrated to obtain a numerical value corresponding to the average
intensity of the signal. The software used for signal analysis is the GEMTOOLS gene
expression analysis program (Incyte).
VIII. Complementary Polynucleotides
[0268] Sequences complementary to the SYNT-encoding sequences, or any parts thereof, are
used to detect, decrease, or inhibit expression of naturally occurring SYNT. Although
use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially
the same procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and
the coding sequence of SYNT. To inhibit transcription, a complementary oligonucleotide
is designed from the most unique 5'sequence and used to prevent promoter binding to
the coding sequence. To inhibit translation, a complementary oligonucleotide is designed
to prevent ribosomal binding to the SYNT-encoding transcript.
IX. Expression of SYNT
[0269] Expression and purification of SYNT is achieved using bacterial or virus-based expression
systems. For expression of SYNT in bacteria, cDNA is subcloned into an appropriate
vector containing an antibiotic resistance gene and an inducible promoter that directs
high levels of cDNA transcription. Examples of such promoters include, but are not
limited to, the
trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the
lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial
hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express SYNT upon induction
with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of SYNT in eukaryotic
cells is achieved by infecting insect or mammalian cell lines with recombinant
Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential
polyhedrin gene of baculovirus is replaced with cDNA encoding SYNT by either homologous
recombination or bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is maintained and the strong polyhedrin promoter drives high levels
of cDNA transcription. Recombinant baculovirus is used to infect
Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection
of the latter requires additional genetic modifications to baculovirus. (See Engelhard.
E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0270] In most expression systems, SYNT is synthesized as a fusion protein with, e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid,
single-step, affinity-based purification of recombinant fusion protein from crude
cell lysates. GST, a 26-kilodalton enzyme from
Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions
that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following
purification, the GST moiety can be proteolytically cleaved from SYNT at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification
on metal-chelate resins (QIAGEN). Methods for protein expression and purification
are discussed in Ausubel (1995,
supra, ch. 10 and 16). Purified SYNT obtained by these methods can be used directly in the
assays shown in Examples X and XIV.
X. Demonstration of SYNT Activity
[0271] An SYNT activity assay measures aminoacylation of tRNA in the presence of a radiolabeled
substrate. A cell-free extract depleted of endogenous aminoacyl-tRNA synthetase is
prepared from
Escherichia coli. SYNT, either biochemically purified or recombinantly produced, is added to the cell
free extract. The cell-free extract is incubated with [
14C]-labeled amino acid under conditions favorable for translation. Incorporation of
the [
14C]-labeled amino acid into acid-precipitable aminoacyl-tRNA is measured using a radioisotope
counter. The amount of the [
14C]-labeled amino acid incorporated into aminoacyl tRNA is proportional to the amount
of SYNT activity. (See, for example. Ibba. M. et al. (1997) Science 278:1119-1 122).
[0272] Alternatively, SYNT activity may be assayed as follows. SYNT, or biologically active
fragments thereof, are labeled with
125I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate
molecules previously arrayed in the wells of a multi-well plate are incubated with
the labeled SYNT, washed, and any wells with labeled SYNT complex are assayed. Data
obtained using different concentrations of SYNT are used to calculate values for the
number, affinity, and association of SYNT with the candidate molecules.
XI. Functional Assays
[0273] SYNT function is assessed by expressing the sequences encoding SYNT at physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian
expression vector containing a strong promoter that drives high levels of cDNA expression.
Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 /µg of recombinant
vector are transiently transfected into a human cell line, for example, an endothelial
or hematopoietic cell line, using either liposome formulations or electroporation.
1-2 µg of an additional plasmid containing sequences encoding a marker protein are
co-transfected. Expression of a marker protein provides a means to distinguish transfected
cells from nontransfected cells and is a reliable predictor of cDNA expression from
the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM),
an automated, laser optics-based technique, is used to identify transfected cells
expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other
cellular properties. FCM detects and quantifies the uptake of fluorescent molecules
that diagnose events preceding or coincident with cell death. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree
side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins as measured
by reactivity with specific antibodies; and alterations in plasma membrane composition
as measured by the binding of fluorescein-conjugated Annexin V protein to the cell
surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994)
Flow Cytometry, Oxford, New York NY.
[0274] The influence of SYNT on gene expression can be assessed using highly purified populations
of cells transfected with sequences encoding SYNT and either CD64 or CD64-GFP. CD64
and CD64-GFP are expressed on the surface of transfected cells and bind to conserved
regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated
from nontransfected cells using magnetic beads coated with either human IgG or antibody
against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods
well known by those of skill in the art. Expression of mRNA encoding SYNT and other
genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of SYNT Specific Antibodies
[0275] SYNT substantially purified using polyacrylamide gel electrophoresis (PAGE; see,
e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
[0276] Alternatively, the SYNT amino acid sequence is analyzed using LASERGENE software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide
is synthesized and used to raise antibodies by means known to those of skill in the
art. Methods for selection of appropriate epitopes, such as those near the C-terminus
or in hydrophilic regions are well described in the art. (See. e.g.. Ausubel, 1995,
supra, ch. 11.)
[0277] Typically, oligopeptides of about 15 residues in length are synthesized using an
ABI 431A peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide
ester (MBS) to increase immunogenicity. (See. e.g., Ausubel, 1995,
supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
Resulting antisera are tested for antipeptide and anti-SYNT activity by, for example,
binding the peptide or SYNT to a substrate, blocking with 1% BSA. reacting with rabbit
antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring SYNT Using Specific Antibodies
[0278] Naturally occurring or recombinant SYNT is substantially purified by immunoaffinity
chromatography using antibodies specific for SYNT. An immunoaffinity column is constructed
by covalently coupling anti-SYNT antibody to an activated chromatographic resin, such
as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is blocked and washed according to the manufacturer's instructions.
[0279] Media containing SYNT are passed over the immunoaffinity column, and the column is
washed under conditions that allow the preferential absorbance of SYNT (e.g., high
ionic strength buffers in the presence of detergent). The column is eluted under conditions
that disrupt antibody/SYNT binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such as urea or thiocyanate ion), and SYNT is collected.
XIV. Identification of Molecules Which Interact with SYNT
[0280] SYNT, or biologically active fragments thereof, are labeled with
125I Bolton-Hunter reagent. (See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J.
133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well
plate are incubated with the labeled SYNT, washed, and any wells with labeled SYNT
complex are assayed. Data obtained using different concentrations of SYNT are used
to calculate values for the number, affinity, and association of SYNT with the candidate
molecules.
[0281] Alternatively, molecules interacting with SYNT are analyzed using the yeast two-hybrid
system as described in Fields, S. and O. Song (1989. Nature 340:245-246), or using
commercially available kits based on the two-hybrid system, such as the MATCHMAKER
system (Clontech).
[0282] SYNT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which
employs the yeast two-hybrid system in a high-throughput manner to determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan. K. et al.
(2000) U.S. Patent No. 6,057,101).